( A ) graphs depict Aβ profiles generated by WT or mutant PSEN1 MEF lines transduced with adenovirus encoding APPC99 . ELISA quantified secreted Aβ37 , Aβ38 , Aβ40 Aβ42 peptide are shown as % of total Aβ ( sum of four peptides ) . blue , orange , green purple dotted lines indicate of Aβ37 , Aβ38 , Aβ40 Aβ42 peptides in WT Aβ profile , respectively . Data are presented as mean ± SD , N ≥ 3 independent experiments .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The graphs depict the Aβ profiles generated by WT or mutant PSEN1 MEF cell lines transduced with adenovirus encoding APPC99. ELISA quantified secreted Aβ37, Aβ38, Aβ40 and Aβ42 peptide levels are shown as % of total Aβ (sum of the four peptides). The blue, orange, green and purple dotted lines indicate the levels of Aβ37, Aβ38, Aβ40 and Aβ42 peptides in the WT Aβ profile, respectively. Data are presented as mean ± SD, N ≥ 3 independent experiments.


( B ) GSEC processivity for cleavage of APPC99 determined in WT mutant PSEN1 / GSEC lines . mutant Aβ ( 37 + 38 ) / ( 40 + 42 ) ratios are shown as % of WT . Data presented as mean ± SD , N ≥ 3 independent experiments . As reference , a dotted was drawn to indicate 10 % ( WT ) . One - way ANOVA followed by Dunnett ' s post - hoc with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) ; * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) GSEC processivity levels for the cleavage of APPC99 determined in WT and mutant PSEN1/GSEC cell lines. The mutant Aβ(37+38)/(40+42) ratios are shown as % of the WT cell line. Data presented as mean ± SD, N ≥ 3 independent experiments. As reference, a dotted line was drawn to indicate 10% (WT levels). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05); **P < 0.01, ***P < 0.001, ****P < 0.0001.


( A ) Aβ profiles generated by WT or mutant PSEN1 MEFs lines treated with 1 µM GSM III . chemical structure of bicyclic heterocycle GSM III is depicted . Data are presented as mean ± SD , N ≥ 3 independent experiments . blue , orange , green purple dotted lines indicate of Aβ37 , Aβ38 , Aβ40 Aβ42 peptides in WT Aβ profile , respectively .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Aβ profiles generated by WT or mutant PSEN1 MEFs lines treated with 1 µM GSM III. The chemical structure of the bicyclic heterocycle GSM III is depicted. Data are presented as mean ± SD, N ≥ 3 independent experiments. The blue, orange, green and purple dotted lines indicate the levels of Aβ37, Aβ38, Aβ40 and Aβ42 peptides in the WT Aβ profile, respectively.


( B ) Fold change in GSEC processivity of APPC99 estimated by Aβ ( 37 + 38 ) / ( 40 + 42 ) presented as % of WT response . data are presented as mean ± SD , N ≥ 3 . symbol # marks mutations that fully abolished GSEC response towards GSM III , As reference , a dotted was drawn to indicate 10 % ( WT ) . One - way ANOVA followed by Dunnett ' s post - hoc with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * * P < 0 . 0001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 32 , 101 ) = 112 . 5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Fold change in GSEC processivity of APPC99 estimated by the Aβ(37+38)/(40+42) ratio and presented as % of the WT response. The data are presented as mean ± SD, N ≥ 3. The symbol # marks mutations that fully abolished the GSEC response towards GSM III, As reference, a dotted line was drawn to indicate 10% (WT levels). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). **P < 0.0001, ****P < 0.0001 (F(DFn, DFd): F (32, 101) = 112.5.


( D ) Aβ profiles generated by WT or mutant PSEN1 lines overexpressing APPC99 treated with increasing concentrations of GSM III . Data are presented as mean ± SD , N = 2 independent experiments .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Aβ profiles generated by WT or mutant PSEN1 cell lines overexpressing APPC99 and treated with increasing concentrations of GSM III. Data are presented as mean ± SD, N = 2 independent experiments.


( E ) GSEC processivity estimated by Aβ ( 37 + 38 ) / ( 40 + 42 ) was determined for dose - response ( presented in panel D ) shown as % of DMSO . Data are presented as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) GSEC processivity estimated by the Aβ(37+38)/(40+42) ratio was determined for the dose-response analysis (presented in panel D) and shown as % of DMSO. Data are presented as mean ± SD.


( A ) Aβ profiles generated by WT indicated mutant MEF lines in presence of vehicle ( 0 . 1 % DMSO ) , 1 µM GSM III Data are presented as mean ± SD , N ≥ 3 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * P < 0 . 01 , * * P < 0 . 0001 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 3 , 45 ) = 56 . 9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle (0.1% DMSO), 1 µM GSM III Data are presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.


( B ) WT mutant GSEC processivity estimated by Aβ ( 37 + 38 ) / ( 40 + 42 ) presented as % of WT . Data presented as mean ± SD , N ≥ 3 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * P < 0 . 01 , * * P < 0 . 0001 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 3 , 45 ) = 56 . 9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) WT and mutant GSEC processivity estimated by the Aβ (37+38)/(40+42) ratio and presented as % of WT. Data presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.


Aβ profiles generated by WT indicated mutant MEF lines in presence of vehicle ( 0 . 1 % DMSO ) , 1 µM GSM III or ( C ) 1 µM GSM II . Data are presented as mean ± SD , N ≥ 3 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * P < 0 . 01 , * * P < 0 . 0001 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 3 , 45 ) = 56 . 9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle (0.1% DMSO), 1 µM GSM III or (C) 1 µM GSM II. Data are presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.


of mutant APP substrates with regards to their effects on response of GSEC to imidazole - GSM III . Aβ profiles secreted by HEK293T transiently expressing WT or mutant APPC99 substrates in presence of vehicle ( DMSO ) or 1 µM GSM III were analysed by ELISA ( D ) ELISA - Aβ profiles are presented as % of total Aβ ( Aβ37 + Aβ38 + Aβ40 + Aβ42 ) ( mean ± SD , N ≥ 3 ) ; One - way ANOVA followed by Dunnett ' s post - hoc with comparison to WT was used to determine statistical significance ; * * * * P < 0 . 0001 . F ( DFn , DFd ) : F ( 4 , 17 ) = 71 . 68 .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole-based GSM III. Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle (DMSO) or 1 µM GSM III were analysed by ELISA (D) ELISA-based Aβ profiles are presented as % of total Aβ (Aβ37+ Aβ38+ Aβ40+ Aβ42) (mean ± SD, N ≥ 3); One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance; ****P < 0.0001. F(DFn, DFd): F (4, 17) = 71.68.


of mutant APP substrates with regards to their effects on response of GSEC to imidazole - GSM III . Aβ profiles secreted by HEK293T transiently expressing WT or mutant APPC99 substrates in presence of vehicle ( DMSO ) or 1 µM GSM III were analysed by mass spectrometry ( MS ) . ( E ) APP mutation - driven effects on GSEC processivity according to Aβ ( 37 + 38 ) / ( 40 + 42 ) are presented as % of WT condition ( mean ± SD ) . One - way ANOVA followed by Dunnett ' s post - hoc with comparison to WT was used to determine statistical significance ; * * * * P < 0 . 0001 . F ( DFn , DFd ) : F ( 4 , 17 ) = 71 . 68 .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole-based GSM III. Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle (DMSO) or 1 µM GSM III were analysed by mass spectrometry (MS). (E) APP mutation-driven effects on GSEC processivity according to the Aβ(37+38)/(40+42) ratio are presented as % of the WT condition (mean ± SD). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance; ****P < 0.0001. F(DFn, DFd): F (4, 17) = 71.68.


( F ) MS - profiles show proportion of Aβ peptides on total immunoprecipitated pool . Data shown as mean of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O

(F) MS-based profiles show the proportion of individual Aβ peptides on the total immunoprecipitated pool. Data shown as mean of 3 independent experiments.


( B ) Aβ profiles generated by WT or mutant PSEN1 lines transduced with APPC99 . Dotted lines indicate of Aβ peptides ( % ) in WT profiles . Data presented as mean ± SD , N ≥ 3 independent experiments .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Aβ profiles generated by WT or mutant PSEN1 cell lines transduced with APPC99. Dotted lines indicate the levels of Aβ peptides (%) in WT profiles. Data presented as mean ± SD, N ≥ 3 independent experiments.


( C ) Pocket filling PSEN1 V236W Y106W - V236W substitutions lead to increased Aβ ( 37 + 38 ) / ( 40 + 42 ) , indicating activation of sequential GSEC - mediated cleavage of APPC99 . Data presented as mean ± SD , N ≥ 3 independent experiments .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Pocket filling PSEN1 V236W and Y106W-V236W substitutions lead to increased Aβ(37+38)/(40+42) ratio, indicating the activation of the sequential GSEC-mediated cleavage of APPC99. Data presented as mean ± SD, N ≥ 3 independent experiments.


( D ) Aβ profile in dose - response experiments for GSM III revealed no modulation of PSEN1 V236W mutant . Data are presented as mean ± SD , N = 2 independent experiments . For comparison purposes , WT V236W mutant Aβ profiles are presented in panel 6B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Aβ profile analysis in dose-response experiments for GSM III revealed no modulation of the PSEN1 V236W mutant cell line. Data are presented as mean ± SD, N = 2 independent experiments. For comparison purposes, WT and V236W mutant Aβ profiles are presented in panel 6B.


( E ) GSEC activity using purified GSEC an APP - fluorogenic peptide as substrate Upper panel : In non - state Dpn ( 2 , 4 - dinitrophenyl ) quencher group suppresses emission from Nma ( N - methyl - o - aminobenzoic acid ) fluorophore through Förster resonance energy transfer ( FRET ) . Upon GSEC cleavage , Nma group emits at λ = 430 nm a signal upon excitation at λ = 355 nm . Lower panel : Specific activities for purified WT or mutant GSECs ( bearing indicated substitutions in PSEN1 ) treated with DMSO ( vehicle ) , 10 µM GSMIII or GSM II . Data are normalized to WT GSEC activity in DMSO ( vehicle ) . Data are shown as mean ± SD , N ≥ 3 independent experiments . Significance is shown for meaningful comparisons ; two - way ANOVA with comparison to WT was used to determine statistical significance ; * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) GSEC activity assay using purified GSEC and an APP-based fluorogenic peptide as substrate Upper panel: In the non-cleaved state the Dpn (2,4-dinitrophenyl) quencher group suppresses fluorescent emission from the Nma (N-methyl-o-aminobenzoic acid) fluorophore through Förster resonance energy transfer (FRET). Upon GSEC cleavage, the Nma group emits at λ = 430 nm a fluorescent signal upon excitation at λ = 355 nm. Lower panel: Specific activities for the purified WT or mutant GSECs (bearing the indicated substitutions in PSEN1) treated with DMSO (vehicle), 10 µM GSMIII or GSM II. Data are normalized to the WT GSEC activity in DMSO (vehicle). Data are shown as mean ± SD, N ≥ 3 independent experiments. Significance is shown for meaningful comparisons; two-way ANOVA with comparison to WT was used to determine statistical significance; **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.


( F ) Transition state analogue inhibitor L - 685 , 458 ( Inhibitor X ) treatment of WT , V236W or Y240W mutant PSEN1 lines . Inhibitory profiles derived IC50 values ( table ) indicate that PSEN1 - V236W significantly increases affinity for inhibitor . Dotted indicates 50 % inhibition . Data presented as mean ± SD , N = 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Transition state analogue inhibitor L-685,458 (Inhibitor X) treatment of WT, V236W or Y240W mutant PSEN1 cell lines. Inhibitory profiles and derived IC50 values (table) indicate that the PSEN1-V236W significantly increases the affinity for the inhibitor. Dotted line indicates 50% inhibition. Data presented as mean ± SD, N = 3 independent experiments.


( H ) Ang - 2 dose time - dependently activates p - EphrinB , p - TIE2 p - AKT in HUVEC ; immunoblotting results ; NA3VO4 : sodium orthovanadate .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O

(H) Ang-2 dose and time-dependently activates p-EphrinB, p-TIE2 and p-AKT in HUVEC; immunoblotting results; NA3VO4: sodium orthovanadate.


( I ) vessels in B16F10 tumors are mostly p - TIE2 ( Tyr992 ) + pSHP2 ( Tyr542 ) + ; confocal ( scale bars : 200μm ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(I) Tumor vessels in B16F10 tumors are mostly p-TIE2 (Tyr992)+ and pSHP2(Tyr542)+; confocal images (scale bars: 200μm).


( A ) Representative tissue immunostaining of human melanoma , colon carcinoma colon adenoma ; confocal from serial sections ( scale bars : 50μm tissues ) . Arrowheads point to CD31 + / p - EphrinB + ; CD31 + / p - SHP2 + ; CD31 + / p - Tie2 + CD31 + / Ang2 + endothelial .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O

(A) Representative tissue immunostaining of human melanoma, colon carcinoma and colon adenoma; confocal images from serial sections (scale bars: 50μm tissues). Arrowheads point to CD31+/p-EphrinB+; CD31+/p-SHP2+; CD31+/p-Tie2+ and CD31+/Ang2+ endothelial cells.


( B ) Gene expression in ( black bar ) control ( bar ) ; top panel : melanoma ( GSE7553 , metastatic ; n = 54 ; control skin n = 5 ) ; bottom panel : colon cancer ( TCGA , metastatic ; n = 269 ; control n = 41 . * * P < 0 . 01 , * * * * P < 0 . 0001 ; P values from Mann Whitney U - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Gene expression in tumor (black bar) and control (white bar); top panel: melanoma (GSE7553, primary and metastatic; tumor n=54; control skin n=5); bottom panel: colon cancer (TCGA, primary and metastatic; tumor n=269; control n=41. **P<0.01, ****P<0.0001; P values from Mann Whitney U-test.


( C ) Impact of high low ( 75 % quartile ) mRNA on survival probability of with melanoma ; Kaplan - Meier using TCGA melanoma datasets ( n = 103 ) ; P values from Mann Whitney U - . ( D ) Impact of high low ( median ) mRNA on survival probability of with colon cancer ; Kaplan - Meier using TCGA colon cancer dataset ( n = 262 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Impact of high and low (75% quartile) mRNA levels on the survival probability of patients with primary melanoma; Kaplan-Meier analysis using TCGA primary melanoma datasets (n=103); P values from Mann Whitney U-test. (D) Impact of high and low (median) mRNA levels on the survival probability of patients with colon cancer; Kaplan-Meier analysis using TCGA colon cancer dataset (n=262).


( C , D ) JAK inhibitor Tofacitinib ( Tofa , 50nM ) enhances proliferation of HUVEC BMEC compared to control ( C ) reduces endogenous p - STAT3 in HUVEC BMEC compared to control ( D ) after 72 - hour culture . SHP099 ( 5μM HUVEC ; 20μM BMEC ) reduces proliferation increases p - STAT3 in HUVEC BMEC compared to control ; error bars : ± S . D . Results of immunoblotting are representative of 3 independent blots .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(C,D) The JAK inhibitor Tofacitinib (Tofa, 50nM) enhances the proliferation of HUVEC and BMEC compared to control (C) and reduces endogenous p-STAT3 levels in HUVEC and BMEC compared to control (D) after 72-hour culture. SHP099 (5μM HUVEC; 20μM BMEC) reduces cell proliferation and increases p-STAT3 in HUVEC and BMEC compared to control; error bars: ± S.D. Results of immunoblotting are representative of 3 independent blots.


( E , F ) MAP kinases inhibitor PD098059 ( PD0980 , 10μM ) SHP099 ( 5μM HUVEC ; 20μM BMEC ) inhibit HUVEC BMEC proliferation compared to control ( E ) reduce p - ERK1 / 2 by immunoblotting after 72 - hour culture ( F ) . Results of proliferation in panels C E ( % of control ) from triplicate cultures are representative of 3 - 4 experiments ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ; two - Student ' s t - ; error bars : ± S . D . Results of immunoblotting are representative of 3 independent blots .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E,F) The MAP kinases inhibitor PD098059 (PD0980, 10μM) and SHP099 (5μM HUVEC; 20μM BMEC) inhibit HUVEC and BMEC proliferation compared to control (E) and reduce p-ERK1/2 levels by immunoblotting after 72-hour culture (F). Results of proliferation in panels C and E (% of control) from triplicate cultures are representative of 3-4 experiments; *P<0.05, **P<0.01 ***P<0.001; two-tailed Student's t-test; error bars: ± S.D. Results of immunoblotting are representative of 3 independent blots.


( G , H ) ERK1 / 2 inhibitors AZD6244 Tamnitinib dose - dependently reduce B16F10 proliferation ( G ) ; representative experiment of 3 independent experiments ; results reflect means of 5 replicate / condition ; error bars : ± S . D . ; of endogenous p - STAT3 p - ERK in B16F10 ( representative of 3 blots ) ( H ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O

(G,H) The ERK1/2 inhibitors AZD6244 and Tamnitinib dose-dependently reduce B16F10 cell proliferation (G); representative experiment of 3 independent experiments; the results reflect the means of 5 replicate/condition; error bars: ± S.D.; and levels of endogenous p-STAT3 and p-ERK in B16F10 cells (representative of 3 blots) (H).


( A - C ) SHP099 ( A , 75mg / kg ; B , C : 100mg / kg ) reduces B16F10 growth in syngeneic mice . Panels depict growth from initiation of treatment to endpoint ( left ) ( right ) at endpoint ; no . mice / group : 6 - 11 control ; 7 - 9 SHP099 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) SHP099 (A, 75mg/kg; B, C: 100mg/kg) reduces B16F10 tumor growth in syngeneic mice. Panels depict tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; no. mice/group: 6-11 control; 7-9 SHP099.


( E ) " Vascular sleeves " ( Collagen IV + CD31 - ) identified in a representative SHP099 - treated . Representative confocal from 6 tumors ( scale bar : 50μm ) . ( F ) Reduced CD31 + vascular in SHP099 - treated tumors compared to controls ( n = 6 / group ) ; by ImageJ . ( G ) of Collagen IV + / CD31 + is increased in SHP099 - treated tumors compared to controls ( n = 6 / group ) ; by ImageJ ; Coll : collagen . Data information : Error bars : ± S . D . ; P values from two - Student ' s t - ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) "Vascular sleeves" (Collagen IV+ CD31-) identified in a representative SHP099-treated tumor. Representative confocal images from 6 tumors (scale bar: 50μm). (F) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ. (G) The ratio of Collagen IV+/CD31+ area is increased in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ; Coll: collagen. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( H ) caspase - 3 detection in a CD31 + vessel ( representative of 6 SHP099 - treated tumors ) ; arrowheads point to caspase - 3 + CD31 + . caspase - 3 + is marked by * . Confocal ( scale bar : 10μm ) . ( I ) Increased caspase - 3 in CD31 + from SHP099 - treated tumors compared to controls ( n = 3 / group ) ; at least 100 counted / sample . ( J ) Ki67 + proliferating in representative of 3 SHP099 - treated 3 control tumors ; CD31 identifies vessels ; DAPI identifies nuclei . Confocal ( scale bar : 50μm ) . ( K ) of proliferating within SHP099 control ( n = 4 / group ) tumors by ImageJ . Data information : Error bars : ± S . D . ; P values from two - Student ' s t - ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Cleaved caspase-3 detection in a CD31+ vessel (representative of 6 SHP099-treated tumors); arrowheads point to cleaved caspase-3+CD31+ cells. Cleaved caspase-3+ tumor cell is marked by *. Confocal images (scale bar: 10μm). (I) Increased cleaved caspase-3 fluorescence intensity in CD31+ cells from SHP099-treated tumors compared to controls (n=3/group); at least 100 cells counted/sample. (J) Ki67+ proliferating cells in representative of 3 SHP099-treated and 3 control tumors; CD31 identifies the vessels; DAPI identifies cell nuclei. Confocal images (scale bar: 50μm). (K) Quantification of proliferating cells within SHP099 and control (n=4/group) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( L ) Hypoxic identified Hypoxyprobe ( green ) representative of 3 SHP099 - treated tumors ; confocal ( scale bar : 100μm ) , left ; of hypoxic areas in control ( n = 3 ) SHP099 - treated ( n = 3 ) tumors by ImageJ . Data information : Error bars : ± S . D . ; P values from two - Student ' s t - ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) Hypoxic tumor identified Hypoxyprobe (green) representative of 3 SHP099-treated tumors; confocal images (scale bar: 100μm), left; quantification of hypoxic areas in control (n=3) and SHP099-treated (n=3) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( M ) Representative control ( of 5 ) SHP099 - treated ( of 5 ) B16F10 sections through maximum diameter ; H & E ( scale bar : 10mm ) . ( N ) caspase - 3 + in SHP099 control tumors ( n = 3 / group ) . Data information : Error bars : ± S . D . ; P values from two - Student ' s t - ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Representative control (of 5) and SHP099-treated (of 5) B16F10 tumor sections through the maximum diameter; H&E staining (scale bar: 10mm). (N) Cleaved caspase-3+ fluorescence intensity in SHP099 and control tumors (n=3/group). Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( B ) Extravasation of FITC - dextran ( green ) in tumors established in NSG mice treated with SHP099 ; scale bar ( 50μm ) . Representative from 3 mice / group . ( C ) Reduced CD31 + vascular in SHP099 - treated tumors compared to controls ( n = 5 / group ) ; ImageJ . P values from two - Student ' s t - ; * P < 0 . 05 . Error bars : ± S . D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Extravasation of FITC-dextran (green) in tumors established in NSG mice treated with SHP099; scale bar (50μm). Representative images from 3 mice/group. (C) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=5/group); ImageJ quantification. P values from two-tailed Student's t-test; *P<0.05. Error bars: ± S.D.


E ) SHP099 reduces of control ( p - LKO vector ) SHP2 - depleted ( shSHP2 ) B16F10 tumors compared to untreated controls : p - LKO untreated ( n = 5 ) , p - LKO treated with SHP099 ( n = 9 ) , shSHP2 untreated ( n = 5 ) , shSHP2 treated with SHP099 ( n = 13 ) . P values by of variance with Dunnett ' s multiple comparison ; * * * P < 0 . 001 . ( F ) Persistent depletion of SHP2 in tumors induced by shSHP2 - B16F10 ( n = 5 ) compared to control tumors induced by p - LKO - B16F10 ( n = 5 ) ; tumors removed from mice 18 days after inoculation .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) SHP099 reduces weight of control (p-LKO vector) and SHP2-depleted (shSHP2) B16F10 tumors compared to untreated controls: p-LKO untreated (n=5), p-LKO treated with SHP099 (n=9), shSHP2 untreated (n=5), and shSHP2 treated with SHP099 (n=13). P values by analysis of variance with Dunnett's multiple comparison test; ***P<0.001. (F) Persistent depletion of SHP2 in tumors induced by shSHP2-B16F10 cells (n=5) compared to control tumors induced by p-LKO-B16F10 (n=5); tumors removed from mice 18 days after cell inoculation.


( N ) Representative popliteal lymph nodes from control SHP099 - treated mice with microscopic evidence of metastases ; H & E stain ; scale bars 500 μm ( low magnification ) 20 μm ( high magnification ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N) Representative popliteal lymph nodes from control and SHP099-treated mice with microscopic evidence of metastases; H&E stain; scale bars 500 μm (low magnification) 20 μm (high magnification).


( C , D ) p - TIE2 in vessels from control AMG386 - treated mice ; representative confocal ; arrowheads point to CD31 + / p - TIE2 - endothelial ( scale bars : 50μm ) ( C ) , ( D ) of p - TIE - 2 / CD31 + in control ( n = 3 ) AMG386 treated ( n = 3 ) tumors ( 1 , 200 CD31 + / group ) . P values from two - Student ' s t - ; * * * P < 0 . 001 . ( E , F ) p - EphrinB ( E ) in representative vessels from control ( n = 3 ) AMG386 - treated ( n = 3 ) tumors ; confocal ( E ) , ( scale bar : 50μm ) of p - EphrinB / CD31 + ( F ) ; P values from two - Student ' s t - ; * * * P < 0 . 001 .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(C,D) p-TIE2 in tumor vessels from control and AMG386-treated mice; representative confocal images; arrowheads point to CD31+/p-TIE2- endothelial cells (scale bars: 50μm) (C), and quantification (D) of p-TIE-2/CD31+ in control (n=3) and AMG386 treated (n=3) tumors (1,200 CD31+ cells/group). P values from two-tailed Student's t-test; ***P<0.001. (E,F) p-EphrinB (E) in representative tumor vessels from control (n=3) and AMG386-treated (n=3) tumors; confocal images (E), (scale bar: 50μm) and quantification of p-EphrinB/CD31+ (F); P values from two-tailed Student's t-test; ***P<0.001.


( G ) Vessel perfusion visualized by FITC - dextran ( green ) ; CD31 ( red ) identifies endothelium ; DAPI ( blue ) identifies nuclei ; representative confocal ( scale bar : 50μm ) of 3 mice / group ; arrow heads point to FITC - dextran extravasation ; asterisks to extravasated erythrocytes .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Vessel perfusion visualized by FITC-dextran (green); CD31 (red) identifies the endothelium; DAPI (blue) identifies the nuclei; representative tumor confocal images (scale bar:50μm) of 3 mice/group; arrow heads point to FITC-dextran extravasation; asterisks to extravasated erythrocytes.


( H ) of caspase - 3 in vasculature ( CD31 + ) ; n = 3 tumors / group ; number of CD31 + counted : control ( n = 311 ) ; SHP099 ( n = 425 ) ; AMG386 ( n = 451 ) ; combined SHP099 + AMG386 ( n = 484 ) . Error bars : ± S . D . ; P values by of variance with Dunnett ' s multiple comparison ; * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Quantification of cleaved caspase-3 in the tumor vasculature (CD31+ cells); n=3 tumors/group; number of CD31+ cells counted: control (n=311); SHP099 (n=425); AMG386 (n=451); combined SHP099+AMG386 (n=484). Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.


( A - D ) p - STAT3 ( A , B ) p - ERK1 / 2 ( C , D ) in CD31 + endothelial of mice treated for 14 days with SHP099 ( 200 mg / kg ) , AMG386 ( 5 . 6mg / kg ) or combination of SHP099 ( 200 mg / kg ) + AMG386 ( 5 . 6mg / kg ) . Representative ( 3 tumors / group ) immunostaining ( A , C ) of / CD31 + ( B , D ) ; horizontal : mean / group ; n = 3 tumors / group ; number of CD31 + counted in B : control ( n = 180 ) , SHP099 ( n = 308 ) , AMG386 ( n = 300 ) , combined SHP099 + AMG386 ( n = 322 ) ; number of CD31 + counted in D : control ( n = 1 , 733 ) , SHP099 ( n = 821 ) , AMG386 ( n = 1 , 399 ) , combined SHP099 + AMG386 ( n = 783 ) . Scale bars : A , 50μm ; C , 10μm .

O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-D) Nuclear p-STAT3 (A,B) and p-ERK1/2 (C,D) in tumor CD31+ endothelial cells of mice treated for 14 days with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) immunostaining (A,C) and quantification of staining intensity/CD31+ cell (B, D); horizontal line: mean intensity/group; n=3 tumors/group; number of CD31+ cells counted in B: control (n=180), SHP099 (n=308), AMG386 (n=300), combined SHP099+AMG386 (n=322); number of CD31+ cells counted in D: control (n=1,733), SHP099 (n=821), AMG386 (n=1,399), combined SHP099+AMG386 (n=783). Scale bars: A, 50μm; C, 10μm.


FOXO1 detection in CD31 + endothelial after treatment with SHP099 ( 200 mg / kg ) , AMG386 ( 5 . 6mg / kg ) or combination of SHP099 ( 200 mg / kg ) + AMG386 ( 5 . 6mg / kg ) . Representative ( 3 tumors / group ) confocal

B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FOXO1 detection in tumor CD31+ endothelial cells after treatment with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) confocal images


( C ) Vessel perfusion visualized by FITC - dextran ( green ) ; CD31 identifies endothelial ( red ) ; DAPI ( blue ) identifies nuclei ; representative ( of 3 mice / group ) confocal ( scale bar : 50μm ) ; arrow heads point to FITC - dextran extravasation .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Vessel perfusion visualized by FITC-dextran (green); CD31 identifies endothelial cells (red); DAPI (blue) identifies the nuclei; representative (of 3 mice/group) confocal images (scale bar: 50μm); arrow heads point to FITC-dextran extravasation.


( H ) Representative depicting Ki67 + in control treated MC38 tumors ; representative ( scale bar : 50μm ) of 4 tumors / group .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(H) Representative images depicting Ki67+ cells in control and treated MC38 tumors; representative images (scale bar: 50μm) of 4 tumors/group.


( A ) WT CITK - / - embryonic ( E14 . 5 ) mousecerebral cortex was stained for γ - tubulin ( green ) ( gray ) . ventricular plane is marked by a red dashed , spindle axis of apical progenitors is indicated by a dashed . angle between these two lines represents mitotic angle . Scale bars = 5 μm . ( B ) of vertical divisions of apical progenitors in WT CITK - / - mice . n = 3 per each genotype .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(A) WT and CITK -/- embryonic (E14.5) mousecerebral cortex was stained for γ-tubulin (green) and DNA (gray). The ventricular plane is marked by a red dashed line, and the spindle axis of apical progenitors is indicated by a white dashed line. The angle between these two lines represents the mitotic angle. Scale bars = 5 μm.(B) Quantification of vertical divisions of apical progenitors in WT and CITK -/- mice. n= 3 per each genotype.


( C ) Pregnant CITK + / - , crossed with CITK + / - , were injected with BrdU at E13 . 5 ; 24 hours later ( E14 . 5 ) WT CITK - / - embryonic mouse cerebral cortex were fixed in 4 % PFA , cryosectioned stained for Ki67 ( red ) , BrdU ( green ) , Tbr2 ( blue ) DAPI ( gray ) . different cortical regions are indicated : CP = cortical plate ; IZ = intermediate zone ; SVZ = subventricular zone ; VZ = ventricular zone . Scale bars = 10 μm . arrows in inset indicate two Ki67 - positive for BrdU Tbr2 .

O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O

(C) Pregnant CITK +/- females, crossed with CITK +/- males, were injected with BrdU at E13.5; 24 hours later (E14.5) WT and CITK -/- embryonic mouse cerebral cortex were fixed in 4% PFA, cryosectioned and stained for Ki67 (red), BrdU (green), Tbr2 (blue) and DAPI (gray). The different cortical regions are indicated: CP=cortical plate; IZ= intermediate zone; SVZ= subventricular zone; VZ= ventricular zone. Scale bars = 10 μm. White arrows in the inset indicate two Ki67- cells positive for BrdU and Tbr2.


( D ) of cycle exit ( of BrdU + / Ki67 − ) in proliferative regions ( VZ / SVZ ) in neuronal layers ( IZ CP ) of sections prepared as in panel C ( n = 4 per each genotype ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of cell cycle exit (ratio of cells BrdU+/ Ki67−) in the proliferative regions (VZ/SVZ) and in the neuronal layers (IZ and CP) of sections prepared as in panel C (n=4 per each genotype).


( E ) Sections prepared as in C were stained for TUBB3 ( TuJ ) to reveal post - mitotic neurons with DAPI . Red arrowheads indicate apoptotic neurons ( defined by pyknotic nuclei ) . of neuronal distribution in VZ - SVZ versus total neurons population is showed on right . Scale bars = 10 μm .

O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Sections prepared as in C were stained for TUBB3 (TuJ) to reveal post-mitotic neurons and with DAPI. Red arrowheads indicate apoptotic neurons (defined by pyknotic nuclei). Quantification of neuronal distribution in VZ-SVZ versus total neurons population is showed on the right. Scale bars = 10 μm.


( F ) of of Tbr2 - positive in BrdU - positive population of same sections analyzed for panel D ( n = 4 per each genotype ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(F) Quantification of the percentage of Tbr2-positive cells in the BrdU-positive population of the same sections analyzed for panel D (n=4 per each genotype).


( G ) Neuroblasts of wild type dck alleles immunostained for α - tubulin Miranda . Note that whereas in wild type there is a tight coupling of mitotic spindle with polarity axis , in dck alleles spindle show a more oblique orientation respect to Mira crescent . Scale bar = 5 μm . ( H ) Distribution of spindle angle amplitude ( ° ) in neuroblasts of wild type ck alleles ( n = 58 ) .

O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Neuroblasts of wild type and dck alleles immunostained for α-tubulin and Miranda. Note that whereas in wild type cells there is a tight coupling of the mitotic spindle with the polarity axis, in dck alleles the spindle show a more oblique orientation respect to Mira crescent. Scale bar = 5 μm.(H) Distribution of spindle angle amplitude (°) in neuroblasts of wild type and ck alleles (n=58).


( A ) Selected frames from time - lapse imaging experiments ( see Movies EV1 EV2 ) showing two dividing transfected with either control ( CTRL ) or CITK - specific siRNAs , respectively . Note one of two daughter in lower panel ( red arrow ) going out of focus as a consequence of oblique division . ( B ) Graphical representation of vertical oblique divisions . cleavage plane is indicated by a black dashed . ( C ) of divisions showing uneven timing of daughter flattening onto substrate after mitosis ( oblique division ) in CITK siRNA treated HeLa compared to control ( n > 50 , 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(A) Selected frames from time-lapse imaging experiments (see Movies EV1 and EV2) showing two dividing cells transfected with either control (CTRL) or CITK-specific siRNAs, respectively. Note one of the two daughter cells in the lower panel (red arrow) going out of focus as a consequence of oblique division.(B) Graphical representation of vertical and oblique divisions. The cleavage plane is indicated by a black dashed line.(C) Quantification of divisions showing uneven timing of daughter cell flattening onto the substrate after mitosis (oblique division) in CITK siRNA treated HeLa cells compared to control (n > 50 cells, 3 independent experiments).


( D ) Control or CITK - depleted were immunostained for γ - tubulin ( red ) ( blue ) imaged in z ( 0 . 3 µm - thick sections ) . Upper panel : maximum projections of confocal z - stacks are shown . Lower panel : cross - section ( XZ ) through two poles of same . ( E ) Distribution of spindle angles ( ° ) in control in CITK - depleted . values represent angles between axis crossing two poles of metaphase spindles coverslip . ( n ≥ 150 , 6 independent experiments ) .

O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Control or CITK-depleted cells were immunostained for γ-tubulin (red) and DNA (blue) and imaged in z (0.3 µm-thick sections). Upper panel: maximum intensity projections of confocal z-stacks are shown. Lower panel: cross-section (XZ) through the two poles of the same cell.(E) Distribution of spindle angles (°) in control and in CITK-depleted cells. The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip. (n ≥ 150 cells, 6 independent experiments).


( F ) of spindle angles average in control in depleted of CITK , ASPM or co - depleted of two ( n ≥ 150 , in at least 3 independent experiments ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(F) Quantification of spindle angles average in control cells and in cells depleted of CITK, ASPM or co-depleted of the two proteins (n ≥ 150 cells, in at least 3 independent experiments).


( A ) HeLa pre - extracted for 1 min with 0 . 5 % Triton X - 100 in PHEM buffer immunostained for CITK ( green ) , γ - tubulin ( red ) ( blue ) . Scale bars ,

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

(A) HeLa cells pre-extracted for 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), γ-tubulin (red) and DNA (blue). Scale bars, 10μm


( B ) HeLa expressing CITK - GFP ( green ) from a BAC transgene treated as in ( A ) immunostained for γ - tubulin ( red ) ( blue ) . Scale bars ,

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

(B) HeLa cells expressing CITK-GFP (green) from a BAC transgene treated as in (A) and immunostained for γ-tubulin (red) and DNA (blue). Scale bars, 5μm


( C ) Western blot of centrosome - containing fractions from HeLa , showing that CITK co - purifies with γ ‑ tubulin .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O

(C) Western blot of centrosome-containing fractions from HeLa cells, showing that CITK co-purifies with γ‑tubulin.


( D ) HeLa pre - extracted 1 min with 0 . 5 % Triton X - 100 in PHEM buffer immunostained for CITK ( green ) , ASPM ( red ) ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O

(D) HeLa cells pre-extracted 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), ASPM (red) and DNA (blue).


( E ) HeLa expressing CITK - GFP treated as in ( A ) immunostained for GFP ( green ) , ASPM ( red ) ( blue ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

(E) HeLa cells expressing CITK-GFP treated as in (A) and immunostained for GFP (green), ASPM (red) and DNA (blue).


( F ) Close physical proximity between CITK ASPM , revealed by PLA on ASPM - GFP - expressing HeLa , incubated with GFP CITK antibodies developed with PLA - specific secondary reagents .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

(F) Close physical proximity between CITK and ASPM, revealed by PLA on ASPM-GFP-expressing HeLa cells, incubated with GFP and CITK antibodies and developed with PLA-specific secondary reagents.


( G ) Control or CITK - depleted immunostained for ASPM ( green ) , γ - tubulin ( red ) ( blue ) .

O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

(G) Control or CITK-depleted cells immunostained for ASPM (green), γ-tubulin (red) and DNA (blue).


( H ) Control or ASPM - depleted treated as in ( A ) immunostained for CITK ( green ) , γ - tubulin ( red ) ( blue ) . ( I ) of CITK positive centrosomes of of CITKspindle pole versus total mean , in control ASPM - depleted . ( n ≥ 75 , 4 independent experiments ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(H) Control or ASPM-depleted cells treated as in (A) and immunostained for CITK (green), γ-tubulin (red) and DNA (blue).(I) Quantification of CITK positive centrosomes and of the ratio of CITKspindle pole intensity versus total cell mean intensity, in control and ASPM-depleted cells. (n ≥ 75 cells, 4 independent experiments).


( A ) Control , CITK or ASPM - depleted HeLa immunostained for α - tubulin ( red ) ( blue ) . Maximum projections of confocal z - stacks are shown . ( B ) of astral MT number maximal in HeLa transfected with CTRL , CITK or ASPM siRNA ( n > 130 in 3 independent experiments ) .

O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O

(A) Control, CITK or ASPM-depleted HeLa cells immunostained for α-tubulin (red) and DNA (blue). Maximum intensity projections of confocal z-stacks are shown.(B) Quantification of astral MT number and maximal length in HeLa cells transfected with CTRL, CITK or ASPM siRNA (n > 130 cells in 3 independent experiments).


( C ) of rescue of astral MT number after CITK overexpression in ASPM - depleted HeLa ( n > 50 in 3 independent experiments ) . ( D ) of spindle orientation rescue after overexpression of CITK , CITN ( lacking kinase domain ) or CKD ( Citron kinase dead ) mutants in ASPM - depleted HeLa ( n > 50 in 3 independent experiments ) .

O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(C) Quantification of the rescue of astral MT number after CITK overexpression in ASPM-depleted HeLa cells (n > 50 cells in 3 independent experiments).(D) Quantification of spindle orientation rescue after overexpression of CITK, CITN (lacking the kinase domain) or CKD (Citron kinase dead) mutants in ASPM-depleted HeLa cells (n > 50 cells in 3 independent experiments).


( E ) Synchronized HeLa transfected with control , ASPM - or CITK - specific siRNAs treated with DMSO or with 250 pM Paclitaxel ( taxol ) 30 min before immunostaining for α - Tubulin . Spindle angles were measured as above ( n > 100 in 6 independent experiments ) .

O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

(E) Synchronized HeLa transfected with control, ASPM- or CITK-specific siRNAs treated with DMSO or with 250 pM Paclitaxel (taxol) 30 min before immunostaining for α-Tubulin. Spindle angles were measured as above (n > 100 cells in 6 independent experiments).


( A ) Exemplar image of EB3 - tdTomatoHeLa used for kymograph analyses . To assess astral MT number dynamics a ( blue ) was added to from pole to cortex .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Exemplar image of EB3-tdTomatoHeLa cell used for kymograph analyses. To assess astral MT number and dynamics a line (blue) was added to images from the pole to the cortex.


( B ) Kymographs of pole ( bottom ) - to - cortex ( top ) scans of control CITK - depleted EB3 - tdTomatoHeLa . maximum projection of a 20 - pixel - wide ( exemplified above ) was plotted at 500 ms time intervals from left to right . MT growth results in diagonal lines .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Kymographs of pole (bottom)-to-cortex (top) line scans of control and CITK-depleted EB3-tdTomatoHeLa cells. The maximum projection of a 20-pixel-wide line (exemplified above) was plotted at 500 ms time intervals from left to right. MT growth results in diagonal lines.


( C ) of astral MT dynamics . MT stability , , growth speed ( n > 250 MT , 30 ) nucleation frequency were calculated ( n > 30 , 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of astral MT dynamics. MT stability, length, growth speed (n > 250 MT, 30 cells) and nucleation frequency were calculated (n > 30 cells, 3 independent experiments).


( D ) Immunofluorescence microscopy showing MT growth at mitotic spindle poles 0 , 1 , 2 min after nocodazole washout . Control CITK - depleted were immunostained for α - tubulin ( red ) ( blue ) . ( E ) of showing detectable MT nucleation ( aster > 1 µm ) 0 , 1 , 2 min after nocodazole washout ( n > 100 , 4 independent experiments ) . ( F ) of aster 2 min after nocodazole washout ( n > 100 , 4 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Immunofluorescence microscopy images showing MT growth at mitotic spindle poles 0, 1, and 2 min after nocodazole washout. Control and CITK-depleted cells were immunostained for α-tubulin (red) and DNA (blue).(E) Percentage of cells showing detectable MT nucleation (aster size > 1 µm) 0, 1, and 2 min after nocodazole washout (n > 100 cells, 4 independent experiments).(F) Quantification of aster size 2 min after nocodazole washout (n > 100 cells, 4 independent experiments).


( A ) activities ( mUnits / g of ) of MRC enzymes were determined by spectrophotometric kinetic measurements in WT ∆ 4 - CYB normalized by of citrate synthase ( CS ) activity . Results are expressed as mean ± SD ( n = 4 - 6 biological replicates ) . Unpaired Student ' s t - * * p = 0 . 0100 ; * * * p = 0 . 0002 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The activities (mUnits/g of protein) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and ∆4-CYB cells and normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=4-6 biological replicates). Unpaired Student's t-test **p=0.0100; ***p=0.0002; ****p<0.0001.


( B ) I in gel activity assays ( IGA ) after Blue - native gel electrophoresis ( BNGE ) of WT ∆ 4 - CYB samples solubilized either with 1 . 6 mg DDM / mg or 4 mg digitonin / mg . gels were incubated in reaction mixture for 1 . 5 hours ( lighter signals in DDM gels ) or were left to continue reaction for 24 hours to obtain darker signals ( DDM Digitonin gels ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Complex I in gel activity assays (IGA) after Blue-native gel electrophoresis (BNGE) of WT and ∆4-CYB samples solubilized either with 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein. The gels were incubated in the reaction mixture for 1.5 hours (lighter signals in DDM gels) or were left to continue the reaction for 24 hours to obtain darker signals (DDM and Digitonin gels).


( C ) BNGE , Western blot immunodetection , with anti - NDUFS1 ( cI ) , anti - ATP5A ( cV ) anti - SDHB ( cII ) antibodies , of samples from WT cybrids from ∆ 4 - CYB clones E ( # 17 . 3E ) B ( # 17 . 3B ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(C) BNGE, Western blot and immunodetection, with anti-NDUFS1 (cI), anti-ATP5A (cV) and anti-SDHB (cII) antibodies, of samples from the WT cybrids and from ∆4-CYB clones E (#17.3E) and B (#17.3B).


( A ) Scatter plot generated from peptide content analyzed by mass spectrometry in each of 64 slices excised from BNGE after quantifying heavy to ( H / L ) ( L / H ) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT ∆ 4 - CYB logarithmic ratios were calculated using MaxQuant statistical significance of differences for enrichment or depletion of were determined with

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

(A) Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy to light (H/L) and (L/H) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and ∆4-CYB cells The logarithmic ratios were calculated using MaxQuant and the statistical significance of the differences for the enrichment or depletion of the proteins were determined with Perseus


( B ) Labeling of thirteen mtDNA - encoded MRC structural subunits . were incubated with [ 35S ] - L - Met for one hour in presence of emetine 100 μg / ml to inhibit translation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Labeling of the thirteen mtDNA-encoded MRC structural subunits. Cells were incubated with [35S]-L-Met for one hour in the presence of emetine 100 μg/ml to inhibit cytoplasmic translation.


( C ) Immunodetection of III structural subunits on Western blots of total lysates separated by SDS - PAGE , from three independent replicates of WT ∆ 4 - CYB . graph shows densitometric of signals corresponding to each subunit normalized to that of β - Actin . mean of three control ( WT ) samples was set to 1 . 0 measurements were referenced to that value . values plotted in graphs are mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons * * * * p < 0 . 0001 ; * * * p = 0 . 0007 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001; ***p=0.0007.


( D ) Immunodetection of I structural subunits on Western blots of total lysates separated by SDS - PAGE , from three independent replicates of WT ∆ 4 - CYB . graph shows densitometric of signals corresponding to each subunit normalized to that of β - Actin . mean of three control ( WT ) samples was set to 1 . 0 measurements were referenced to that value . values plotted in graphs are mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons * * * * p < 0 . 0001 ; * * p = 0 . 0024 ( NDUFS3 ) ; * * p = 0 . 0061 ( NDUFB8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O

(D) Immunodetection of complex I structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001; **p=0.0024 (NDUFS3); **p=0.0061 (NDUFB8).


( E ) Immunodetection of IV structural subunits on Western blots of total lysates separated by SDS - PAGE , from three independent replicates of WT ∆ 4 - CYB . graph shows densitometric of signals corresponding to each subunit normalized to that of β - Actin . mean of three control ( WT ) samples was set to 1 . 0 measurements were referenced to that value . values plotted in graphs are mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons * * p = 0 . 0011 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Immunodetection of complex IV structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0011.


( F ) Immunodetection of II structural subunits on Western blots of total lysates separated by SDS - PAGE , from three independent replicates of WT ∆ 4 - CYB . graph shows densitometric of signals corresponding to each subunit normalized to that of β - Actin . mean of three control ( WT ) samples was set to 1 . 0 measurements were referenced to that value . values plotted in graphs are mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001.


( G ) Immunodetection of V structural subunits on Western blots of total lysates separated by SDS - PAGE , from three independent replicates of WT ∆ 4 - CYB . graph shows densitometric of signals corresponding to each subunit normalized to that of β - Actin . mean of three control ( WT ) samples was set to 1 . 0 measurements were referenced to that value . values plotted in graphs are mean ± SD ( n = 3 ) . There were no differences in steady - state of tested subunits ( 2 - way ANOVA with Sidak ' s multiple comparisons ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Immunodetection of complex V structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). There were no differences in the steady-state levels of the tested subunits (2-way ANOVA with Sidak's multiple comparisons test).


( A ) Complexome profiles of cIII2 structural subunits generated by analyzing peptide content in each of 64 slices in which gel lanes were excised graphs plot relative peptide peak intensities along lane , setting maximum to 1 . 0 , vs . molecular mass calculated using complexes supercomplexes as standards to generate a calibration curve . relative amounts of between two lines was determined by calculating H / L ratios of peptides that were present in both WT ( blue traces ) ∆ 4 - CYB samples ( red traces ) . represented values are mean ± of two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(A) Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0, vs. the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines was determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and ∆4-CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( B ) Second - dimension BNGE of digitonin - solubilized samples from WT ∆ 4 - CYB , western blot immunodetection of indicated cIII2 structural subunits with specific antibodies . immunodetection patterns were equivalent to complexome profiles .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(B) Second-dimension BNGE of digitonin-solubilized samples from WT and ∆4-CYB cells, western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles.


( C ) of total peak under curves ( AUC ) defined by peptide peaks for indicated cIII2 subunits . x - axis values were slice number ( 1 - 64 ) y - axis values were relative peptide . graph shows mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons * * p = 0 . 0083 ( UQCRC2 ) ; * * p = 0 . 0033 ( UQCRFS1 ) ; * p = 0 . 0224 ; n . s . = non - significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(C) Quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cIII2 subunits. The x-axis values were the slice number (1-64) and the y-axis values were the relative peptide intensity. The graph shows the mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0083 (UQCRC2); **p=0.0033 (UQCRFS1); *p=0.0224; n.s.=non-significant.


( D ) Complexome profiles of two cIII2 assembly factors ( BCS1L LYRM7 or MZM1L ) generated represented values are mean ± of two reciprocal labeling experiments .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O

(D) Complexome profiles of two cIII2 assembly factors (BCS1L and LYRM7 or MZM1L) generated The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( A ) SDS - PAGE , Western blot an immunodetection , with indicated specific antibodies , of ∆ 4 - CYB WT expressing HA - tagged versions of UQCRQ UQCR10 of transduced with lentiviral expression vector without any cDNA insert ( Empty ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

(A) SDS-PAGE, Western blot an immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing HA-tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).


( B ) BNGE , Western blot immunodetection , with an anti - HA tag antibody , of samples from same lines as in ( A ) solubilized either with digitonin or DDM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the same cell lines as in (A) solubilized either with digitonin or DDM.


( C ) BNGE , Western blot immunodetection , with monoclonal ( M ) anti - UQCRQ antibody ( Abcam ab110255 ) , of non - transduced ∆ 4 - CYB WT . mitoplast samples were solubilized with

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O

(C) BNGE, Western blot and immunodetection, with the monoclonal (M) anti-UQCRQ antibody (Abcam ab110255), of non-transduced ∆4-CYB and WT cells. The mitoplast samples were solubilized with DDM


( D ) Scatter plot generated from of logarithmic heavy ( H ) to ( L ) ratios in x - axis reverse in y - axis , in two reciprocal labeling SILAC experiments ( 1 2 ) anti - HA immunopurification of ∆ 4 - CYB WT expressing UQCR10HA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

(D) Scatter plot generated from the analysis of the logarithmic heavy (H) to light (L) ratios in the x-axis and the reverse in the y-axis, in the two reciprocal labeling SILAC experiments (1 and 2) and anti-HA immunopurification of ∆4-CYB and WT cells expressing UQCR10HA.


( E ) Complexome profiles for found specifically enriched in ∆ 4 - CYB UQCR10HA , according to SILAC immunopurification experiments represented values are mean ± of two reciprocal labeling experiments .

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

(E) Complexome profiles for the proteins found specifically enriched in ∆4-CYB UQCR10HA, according to the SILAC immunopurification experiments The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( F ) SDS - PAGE , Western blot immunodetection , with indicated specific antibodies , of ∆ 4 - CYB WT expressing an HA - tagged version of CYC1 of transduced with lentiviral expression vector without any cDNA insert ( Empty ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(F) SDS-PAGE, Western blot and immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing an HA-tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).


( G ) BNGE , Western blot immunodetection , with an anti - HA tag antibody , of samples from lines solubilized either with digitonin .

O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the cell lines solubilized either with digitonin.


( H ) Scatter plot generated from of logarithmic heavy ( H ) to ( L ) ratios in x - axis reverse in y - axis , in two reciprocal labeling SILAC experiments of anti - HA immunopurification of ∆ 4 - CYB WT expressing CYC1HA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

(H) Scatter plot generated from the analysis of the logarithmic heavy (H) to light (L) ratios in the x-axis and the reverse in the y-axis, in the two reciprocal labeling SILAC experiments of anti-HA immunopurification of ∆4-CYB and WT cells expressing CYC1HA.


( A ) Complexome profiles of different cI structural modules . graphs were generated but in this case , peptide values for subunits belonging to each module were averaged to simplify represented values are mean ± of two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Complexome profiles of the different cI structural modules. The graphs were generated but in this case, the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( B ) Complexome profiles of cI assembly factor NDUFAF2 in WT ( blue ) ∆ 4 - CYB ( red ) mitochondria represented values are mean ± of two reciprocal labeling experiments .

O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

(B) Complexome profiles of the cI assembly factor NDUFAF2 in WT (blue) and ∆4-CYB (red) mitochondria The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( C ) of total under curve in profiles corresponding to each cI module ( top graph ) . of NDUFA9 subunit was calculated separately as its profile did not correspond to any of other structural subunit modules . plotted values are mean mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons * * * p = 0 . 0006 ; * * p = 0 . 0012 ( N - module ) ; * * p = 0 . 0016 ( ND1 - module ) ; * * p = 0 . 0032 ( ND2 - module ) ; * * p = 0 . 0057 ( ND4 - module ) ; * * p = 0 . 0020 ( ND5 - module ) ; * p = 0 . 0140 .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

(C) Quantification of the total area under the curve in the profiles corresponding to each cI module (top graph). The area of the NDUFA9 subunit was calculated separately as its profile did not correspond to any of the other structural subunit modules. The plotted values are the mean mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test ***p=0.0006; **p=0.0012 (N-module); **p=0.0016 (ND1-module); **p=0.0032 (ND2-module); **p=0.0057 (ND4-module); **p=0.0020 (ND5-module); *p=0.0140.


( D ) of of N - module peak corresponding to molecular mass of fully assembled cI : 1 , 172 kDa in ∆ 4 - CYB samples respirasome ( cI + cIII2 + cIV ) SCs in # 4 . 1 WT samples ( 2 , 110 kDa ) . plotted values are mean mean ± SD ( n = 2 ) . Unpaired Student ' s t - * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of the area of the N-module peak corresponding to the molecular mass of the fully assembled cI: 1,172 kDa in ∆4-CYB samples and the respirasome (cI+cIII2+cIV) SCs in #4.1 WT samples (2,110 kDa). The plotted values are the mean mean ± SD (n=2). Unpaired Student's t-test ****p<0.0001.


( A ) SDS - PAGE resolving radioactively labeled mitochondrial translation products after a 2 - hour ( P ) . 35S - Met cycloheximide were removed from medium were collected at indicated chase times ( 2 , 5 24 hours ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) SDS-PAGE resolving the radioactively labeled mitochondrial translation products after a 2-hour pulse (P). The 35S-Met and the cycloheximide were removed from the medium and cells were collected at the indicated chase times (2, 5 and 24 hours).


( B ) First dimension ( 1D ) BNGE of same prepared with digitonin .

(B) First dimension (1D) BNGE analysis of the same cells prepared with digitonin.


( C ) Denaturing second dimension ( 2D ) BNGE of same samples allowed following incorporation of labeled subunits inside their corresponding supercomplex species .

O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Denaturing second dimension (2D) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species.


( D ) I IGA ( cI - IGA ) of digitonin - solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days ( 0h ) . After removing drug restoring synthesis of mtDNA - encoded subunits , were collected at indicated times to follow appearance of cI reactivity with time . gels were incubated in reaction mixture for 24 hours . SS = steady state . asterisk indicates presence of a high molecular cI - containing band of unknown

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Complex I IGA (cI-IGA) analysis of digitonin-solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days (0h). After removing the drug and restoring synthesis of the mtDNA-encoded subunits, the cells were collected at the indicated times to follow the appearance of cI reactivity with time. The gels were incubated in the reaction mixture for 24 hours. SS = steady state. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature


( E ) 2D BNGE , Western blot immunodetection of WT ∆ 4 - CYB mitochondria from collected at same times after doxycycline treatment to follow incorporation kinetics of indicated cI - encoded subunits , belonging to different structural modules . blots shown were either exposed for 16 sec . ( low exposures ) or 160 sec . ( high exposures ) in order to visualize qualitative signals in ∆ 4 - CYB samples .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O

(E) 2D BNGE, Western blot and immunodetection analysis of WT and ∆4-CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear-encoded subunits, belonging to different structural modules. The blots shown were either exposed for 16 sec. (low exposures) or 160 sec. (high exposures) in order to visualize the qualitative signals in the ∆4-CYB samples.


( A ) SDS - PAGE , Western blot immunodetection of AOXHA expression in whole - lysates from WT ∆ 4 - CYB transduced with AOXHA / pWPXLd - ires - HygroR lentiviral vector . transduction selection controls were same lines transfected with an empty pWPXLd - ires - HygroR vector ( EV ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) SDS-PAGE, Western blot and immunodetection of AOXHA expression in whole-cell lysates from WT and ∆4-CYB cells transduced with the AOXHA/pWPXLd-ires-HygroR lentiviral vector. The transduction and selection controls were the same cell lines transfected with an empty pWPXLd-ires-HygroR vector (EV).


( B ) Growth curves of AOXHA expressing lines their corresponding EV controls . growth was monitored every six hours after substituting medium in two replicate 24 - well plates , one plate with medium without uridine ( Uridine - ) , second plate with medium supplemented with 50 µg / ml uridine ( Uridine + ) . graphs show average confluence ± SD at each time point ( n = 6 wells per ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Growth curves of the AOXHA expressing cell lines and their corresponding EV controls. Cell growth was monitored every six hours after substituting the medium in two replicate 24-well plates, one plate with medium without uridine (Uridine-), and the second plate with medium supplemented with 50 µg/ml uridine (Uridine+). The graphs show the average confluence ± SD at each time point (n=6 wells per cell line).


( C ) Immunodetection of cI structural subunits NDUFAF2 in same

(C) Immunodetection of cI structural subunits and NDUFAF2 in the same samples


( D ) 1D BNGE , Western blot immunodetection analyses of digitonin - solubilized mitochondria from WT ∆ 4 - CYB cybrids expressing AOXHA their corresponding EV controls .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O

(D) 1D BNGE, Western blot and immunodetection analyses of digitonin-solubilized mitochondria from WT and ∆4-CYB cybrids expressing AOXHA and their corresponding EV controls.


( E ) I in gel activity assays ( IGA ) after BNGE gels were incubated in IGA reaction mixture for 5 hours . asterisk indicates presence of a high molecular cI - containing band of unknown

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Complex I in gel activity assays (IGA) after BNGE The gels were incubated in the IGA reaction mixture for 5 hours. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature


( F ) Spectrophotometric kinetic measurements of cI activity in WT ∆ 4 - CYB AOXHA EV samples , normalized by of citrate synthase ( CS ) activity . Results are expressed as mean ± SD ( n = 5 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons * * p = 0 . 0077 ( WT AOXHA vs . ∆ 4 - CYB AOXHA ) ; * * p = 0 . 0044 ( ∆ 4 - CYB EV vs . ∆ 4 - CYB AOXHA ) ; * * * p = 0 . 0003 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(F) Spectrophotometric kinetic measurements of cI activity in WT and ∆4-CYB AOXHA and EV samples, normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=5 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test **p=0.0077 (WT AOXHA vs. ∆4-CYB AOXHA); **p=0.0044 (∆4-CYB EV vs. ∆4-CYB AOXHA); ***p=0.0003; ****p<0.0001.


( G ) High resolution respirometry analyses performed in intact in an Oroboros instrument . ROUTINE : basal oxygen consumption rate ( in pmol O2 / sec ) per million in DMEM medium . LEAK is non - phosphorylating respiration in presence of ATP synthase inhibitor oligomycin . ETS : Maximal respiration rate in presence of uncoupler CCCP . AOX : oxygen consumption in presence of antimycin A , inhibiting cIII2 activity but not that of AOX . ROTENONE : Oxygen consumption in presence of cI inhibitor rotenone . In cases this was equal to background . Results are expressed as mean ± SD ( n = 2 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) High resolution respirometry analyses performed in intact cells in an Oroboros instrument. ROUTINE: cellular basal oxygen consumption rate (in pmol O2/sec) per million cells in DMEM medium. LEAK is the non-phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin. ETS: Maximal respiration rate in the presence of the uncoupler CCCP. AOX: oxygen consumption in the presence of antimycin A, inhibiting cIII2 activity but not that of AOX. ROTENONE: Oxygen consumption in the presence of the cI inhibitor rotenone. In all cases this was equal to the background. Results are expressed as mean ± SD (n=2 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.


( H ) Respirometry analyses , in WT EV controls AOXHA - expressing untreated or treated with 2 . 5 µM antimycin A for 7 days ( + AA ) . Results are expressed as mean ± SD ( n = 4 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons * * * * p < 0 . 0001 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Respirometry analyses , in WT EV controls and AOXHA- expressing cells untreated or treated with 2.5 µM antimycin A for 7 days (+AA). Results are expressed as mean ± SD (n=4 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.


( I ) 1D BNGE followed by cI - IGA ( right ) or Western blot immunodetection of cIII2 subunit CYC1 ( left ) in digitonin - solubilized samples from WT EV controls AOXHA - expressing untreated ( - ) or treated ( + ) with 2 . 5 µM antimycin A for 7 days .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(I) 1D BNGE followed by cI-IGA (right) or Western blot and immunodetection of cIII2 subunit CYC1 (left) in digitonin-solubilized samples from WT EV controls and AOXHA- expressing cells untreated (-) or treated (+) with 2.5 µM antimycin A for 7 days.


( A ) Complexome profiles of two cV structural assembly modules . graphs were generated as in Figure 3 but in this case , peptide values for subunits belonging to each module were averaged to simplify . represented values are mean ± of two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Complexome profiles of the two cV structural and assembly modules. The graphs were generated as in Figure 3 but in this case, the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( B ) Complexome profiles of three detected cII subunits . represented values are mean ± of two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O

(B) Complexome profiles of the three detected cII subunits. The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( C ) Complexome profiles of different cIV assembly modules of last subunit to be incorporated ( NDUFA4 ) . , peptide values for subunits belonging to each assembly module were averaged to simplify . represented values are mean ± of two reciprocal labeling experiments . bar graph represents of total under curve in profiles corresponding to each cIV module . plotted values are mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons * * p = 0 . 0088 ( Early ) ; * * p = 0 . 0063 ( MT - CO2 ) ; * * p = 0 . 0040 ( NDUFA4 ) ; * p = 0 . 0264 .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

(C) Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated (NDUFA4). , the peptide intensity values for the individual subunits belonging to each assembly module were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total area under the curve in the profiles corresponding to each cIV module. The plotted values are mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0088 (Early); **p=0.0063 (MT-CO2); **p=0.0040 (NDUFA4); *p=0.0264.


( D ) 1D BNGE , Western blot immunodetection of two cIV subunits ( MT - CO1 COX7B ) in samples from WT ∆ 4 - CYB solubilized with DDM digitonin ( Dig ) .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

(D) 1D BNGE, Western blot and immunodetection of two cIV subunits (MT-CO1 and COX7B) in samples from WT and ∆4-CYB cells solubilized with DDM and digitonin (Dig).


D ) of zone 1 - 3 uninfected hepatocytes day 5 post plating ( + / - s . d . ) of 4 different donors ( blue dots ) . For each donor , > 500 were characterized for final percentages . Kruskal Wallis ( p = 0 . 0002 ) followed by Dunn ' s multiple comparisons where Z1 / hGK + + / hGS - versus Z2 / hGK + / hGS - has a p value of 0 . 0140 ( other comparisons of Z1 / hGK + + / hGS - vs Z3 / hGK - / hGS + Z2 / hGK + / hGS - vs Z3 / hGK - / hGS + were non - significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Percentage of zone 1-3 uninfected hepatocytes day 5 post plating (+/- s.d.) of 4 different donors (blue dots). For each donor, >500 cells were characterized for the final percentages. Kruskal Wallis test (p = 0.0002) followed by Dunn's multiple comparisons test where Z1/hGK++/hGS- versus Z2/hGK+/hGS- has a p value of 0.0140 (the other comparisons of Z1/hGK++/hGS- vs Z3/hGK-/hGS+ and Z2/hGK+/hGS- vs Z3/hGK-/hGS+ were non-significant).


C : Expression of typical liver stage markers : Circumsporozoite ( CSP ) top left ; Exported 2 ( EXP2 ) top right ; Glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) bottom left ; Merozoite surface 1 ( MSP1 ) bottom right . Scale bar is 25 µm .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O

C: Expression of typical liver stage protein markers: Circumsporozoite protein (CSP) top left; Exported Protein 2 (EXP2) top right; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bottom left; and Merozoite surface protein 1 (MSP1) bottom right. Scale bar is 25 µm.


D ) schizonts ( from total number of schizonts in a well + / - s . d . ) located in different hepatocyte sub - populations by NF54 , NF135 , NF175 on day 3 p . i . . Each dot represents a biological replicate . For each , at least 100 schizonts were characterized from 2x 96 wells for final . values from three biological replicates were used in a RM ANOVA followed by a Tukey ' s multiple comparison . of schizonts in Z1 / hGK + + / hGS - hepatocytes is not different between three strains . of schizonts in Z2 / hGK + / hGS - for NF54 is significantly different from NF135 ( p = 0 . 0015 ) NF175 ( p = 0 . 0015 ) . of schizonts in Z3 / hGK - / hGS + hepatocytes for NF54 is significantly different from NF135 ( p = 0 . 0002 ) NF175 ( p = 0 . 0 . 0002 ) . Data information : * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Percentage schizonts (from total number of schizonts in a well +/- s.d.) located in the different hepatocyte sub-populations by NF54, NF135, NF175 on day 3 p.i.. Each dot represents a biological replicate. For each parasite line, at least 100 schizonts were characterized from 2x 96 wells for the final percentage. The values from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Percentage of schizonts in Z1/hGK++/hGS- hepatocytes is not different between the three parasite strains. Percentage of schizonts in Z2/hGK+/hGS- for NF54 is significantly different from NF135 (p = 0.0015) and NF175 (p = 0.0015). Percentage of schizonts in Z3/hGK-/hGS+ hepatocytes for NF54 is significantly different from NF135 (p = 0.0002) and NF175 (p = 0.0.0002). Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001


F ) of schizonts in Z1 / hGK + + / hGS - , Z2 / hGK + / hGS - Z3 / hGK - / hGS + hepatocytes on day 3 post invasion for different Pf strains . For each , at least 100 schizonts were measured from each biological replicate . median values + / - s . d . ( as sizes within population is not normally distributed ) from three biological replicates were used in a RM ANOVA followed by a Tukey ' s multiple comparison . Data information : * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Size of schizonts in Z1/hGK++/hGS-, Z2/hGK+/hGS- and Z3/hGK-/hGS+ hepatocytes on day 3 post invasion for the different Pf strains. For each parasite line, at least 100 schizonts were measured from each biological replicate. The median values +/- s.d. (as the sizes within the population is not normally distributed) from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001


hGS interaction with NF135 ( A ) , NF175 ( B ) schizonts on days 3 ( top ) , 5 ( middle ) 7 ( bottom ) p . i . . hGS ( green ) , PfHSP70 ( red ) , DAPI ( blue ) phalloidin ( grey ) . U panel is composed an uninfected zone 3 hepatocyte on day 3 p . i . . Scale bar is 25 µm .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hGS interaction with NF135 (A), NF175 (B) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.


hGS interaction with NF54 ( C ) schizonts on days 3 ( top ) , 5 ( middle ) 7 ( bottom ) p . i . . hGS ( green ) , PfHSP70 ( red ) , DAPI ( blue ) phalloidin ( grey ) . U panel is composed an uninfected zone 3 hepatocyte on day 3 p . i . . Scale bar is 25 µm .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hGS interaction with NF54 (C) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.


D ) Median sizes ( + / - s . d . ) of NF135 ( green ) , NF175 ( purple ) NF54 ( blue ) schizonts divided on presence ( solid ) or absence ( dashed ) of hGS pattern on high content microscopy from three different liver donors where each donor has at least 100 schizonts were measured for each of three biological replicates . A two - way RM ANOVA was performed on median followed by a Tukey ' s multiple comparisons . significant differences between hGS + hGS - population within a is showed with asterisks ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . See Appendix Fig S1 for raw measurements .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Median sizes (+/- s.d.) of NF135 (green), NF175 (purple) and NF54 (blue) schizonts divided based on the presence (solid line) or absence (dashed line) of hGS staining pattern based on high content fluorescence microscopy from three different liver donors where each donor has at least 100 schizonts were measured for each of three biological replicates. A two-way RM ANOVA was performed on the median followed by a Tukey's multiple comparisons test. The significant differences between hGS+ and hGS- population within a strain is showed with asterisks (* p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001). See Appendix Fig S1 for the raw measurements.


A ) total hGS signal within intracellular NF135 B ) NF175 schizonts on day 3 5 . Each graph represents a biological replicate where each dot represents an schizont .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

A) The total hGS signal within intracellular NF135 and B) NF175 schizonts on day 3 and 5. Each graph represents a biological replicate where each dot represents an individual schizont.