( D ) RNF114 depletion impaired early embryonic development . Rnf114 siRNA # 1 or siRNA # 2 injected zygotes were cultured quantified ( siRNA1 # , n = 120 ; siRNA2 # , n = 128 ; three independent experiments ) ; NC indicates negative control ( n = 193 ; six independent experiments ) . error bars represent s . d . , * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 in unpaired two - t - .

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(D) RNF114 depletion impaired the early embryonic development. Rnf114 siRNA #1 or siRNA #2 injected zygotes were cultured and quantified (siRNA1#, n = 120; siRNA2#, n = 128; three independent experiments); NC indicates negative control (n = 193; six independent experiments). The error bars represent s.d., *:P<0.05, **:P<0.01, ***:P<0.001 in unpaired two-tailed t-test.


( E ) Representative to show early embryonic development defects . arrested at two - stage are indicated by arrows ( Scale bar = 100um ) .

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(E) Representative images to show early embryonic development defects. The embryos arrested at two-cell stage are indicated by arrows (Scale bar = 100um).


( A ) In vitro ubiquitination . Purified His - RNF114 was subjected to ubiquitination assays with indicated components samples were analyzed by immunoblotting with anti - FLAG ( Ub ) antibody . Lanes 1 - 4 indicate negative controls . results show that recombinant RNF114 efficiently catalyzed formation of polyubiquitin chains .

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(A) In vitro ubiquitination assay. Purified His-RNF114 was subjected to ubiquitination assays with the indicated components and the samples were analyzed by immunoblotting with anti-FLAG (Ub) antibody. Lanes 1-4 indicate negative controls. The results show that recombinant RNF114 efficiently catalyzed the formation of polyubiquitin chains.


( B , C ) Purified wild - type RING mutated His - RNF114 were stained by Coomassie Blue ( B ) . Purified wild - type RING mutated His - RNF114 were subjected to ubiquitination assays together with E1 , Ubc4 , FLAG - Ub , ATP . results show that mutant RNF114 lacked ubiquitination activity ( C ) .

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(B, C) Purified wild-type and RING mutated His-RNF114 were stained by Coomassie Blue (B). Purified wild-type and RING mutated His-RNF114 were subjected to ubiquitination assays together with E1, Ubc4, FLAG-Ub, and ATP. The results show that mutant RNF114 lacked ubiquitination activity (C).


( D ) Screening for ubiquitinated by RNF114 on a proteome array . Putative substrates of RNF114 were identified on Ubiquitin Ligase Substrate Identification on ProtoArray ® Human Microarrays v5 . 0 . Ubiquitination reactions were performed on quintuplicate arrays using indicated components , five sub - array are respectively negative controls , E1 + E2 ( no E3 ) , WT RNF114 ( two arrays , duplicate ) , mutated RNF114 .

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(D) Screening for proteins ubiquitinated by RNF114 on a proteome array. Putative substrates of RNF114 were identified on Ubiquitin Ligase Substrate Identification on ProtoArray® Human Protein Microarrays v5.0. Ubiquitination reactions were performed on quintuplicate arrays using the indicated components, the five sub-array are respectively negative controls, E1+E2 (no E3), WT RNF114 (two arrays, duplicate), and mutated RNF114.


( E ) A heat map comparing hits between E1 + E2 , E1 + E2 + WT RNF114 , E1 + E2 + mutated RNF114 reactions . Candidate substrates of RNF114 selected for further characterization are indicated in red font ; HIP1 , indicated by blue font , was considered to be a false positive because it was also detected in mutated RNF114 reaction . Black font indicates with ubiquitin - binding domains such as UIM , UBA or CUE or without clearly reported functions , which were excluded from further investigation .

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(E) A heat map comparing hits between E1+E2, E1+E2+WT RNF114, and E1+E2+mutated RNF114 reactions. Candidate substrates of RNF114 selected for further characterization are indicated in red font; HIP1, indicated by blue font, was considered to be a false positive because it was also detected in the mutated RNF114 reaction. Black font indicates proteins with ubiquitin-binding domains such as UIM, UBA or CUE or proteins without clearly reported functions, which were excluded from further investigation.


( A ) RNF114 substrate validation . HEK293 were co - transfected with constructs expressing FLAG - tagged substrates , wild - type or mutated His - RNF114 EGFP , western blot was performed with anti - FLAG anti - His antibodies . results show that of CD74 , TAB1 , OPTN , TNIP1 , APPL1 , PSAT1 , RALGPS1 were reduced in presence of wild - type RNF114 compared to co - transfecting with mutant RNF114 .

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(A) RNF114 substrate validation. HEK293 cells were co-transfected with constructs expressing FLAG-tagged substrates, wild-type or mutated His-RNF114 and EGFP, and western blot was performed with anti-FLAG and anti-His antibodies. The results show that the protein levels of CD74, TAB1, OPTN, TNIP1, APPL1, PSAT1, and RALGPS1 were reduced in the presence of wild-type RNF114 compared to co-transfecting with mutant RNF114.


( B ) RNF114 - dependent in vitro ubiquitination of substrates . Substrate ubiquitination assays were performed with purified RNF114 , reaction products were immunoblotted using anti - His antibody to detect ubiquitin ( upper panel ) or anti - FLAG antibody detecting substrate ( middle panel ) , lower panel showed merged signals . results show that clear polyubiquitin chains were formed in systems containing recombinant CD74 , TAB1 , TNIP1 , PSAT1 or RALGPS1 .

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(B) RNF114-dependent in vitro ubiquitination of substrates. Substrate ubiquitination assays were performed with purified RNF114, the reaction products were immunoblotted using anti-His antibody to detect the ubiquitin (upper panel) or anti-FLAG antibody detecting the substrate protein (middle panel), and the lower panel showed the merged signals. The results show that clear polyubiquitin chains were formed in the systems containing recombinant CD74, TAB1, TNIP1, PSAT1 or RALGPS1.


( A ) Mouse zygotes were microinjected with sterile ddH2O ( control group ) or Ralgps1 , Cd74 , Tnip1 , Psat1 , or Tab1 mRNA . During 96 - 100 h culturing , percentages of at various stages were counted under microscopy . results show that overexpression of Tab1 impaired early embryonic development . Three independent experiment replicates were performed , error bars represent s . d . ; * : P < 0 . 05 , * * * : P < 0 . 001 in unpaired two - t - .

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(A) Mouse zygotes were microinjected with sterile ddH2O (control group) or Ralgps1, Cd74, Tnip1, Psat1, or Tab1 mRNA. During 96-100 h culturing, the percentages of embryos at various stages were counted under microscopy. The results show that overexpression of Tab1 impaired the early embryonic development. Three independent experiment replicates were performed, error bars represent s.d.; *:P<0.05, ***:P<0.001 in unpaired two-tailed t-test.


( B ) TAB1 reduced with mouse early embryonic development , while abnormally accumulated at 2 - stage once treated with Rnf114 siRNAs . TAB1 was detected by western blotting with anti - TAB1 antibody . relative level of TAB1 after Rnf114 siRNAs treatment were presented as mean ± s . e . m . ( * : P < 0 . 05 in unpaired two - t - , four independent experiment replicates ) .

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(B) TAB1 reduced with mouse early embryonic development, while abnormally accumulated at 2-cell stage once treated with Rnf114 siRNAs. TAB1 was detected by western blotting with anti-TAB1 antibody. The relative level of TAB1 protein after Rnf114 siRNAs treatment were presented as mean±s.e.m. (*:P<0.05 in unpaired two-tailed t-test, four independent experiment replicates).


( C ) Detection of endogenous TAB1 ubiquitination at 1 - or 2 - stages . Endogenous TAB1 were immunoprecipated by anti - TAB1 antibody at either 1 - or 2 - stages , immunoprecipates were detected by either anti - TAB1 antibody or anti - ubiquitin antibody . results show that more Ub chains on TAB1 were detected in two stage than in zygotes , when TAB1 were at comparable level .

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(C) Detection of endogenous TAB1 ubiquitination at 1-cell or 2-cell stages. Endogenous TAB1 proteins were immunoprecipated by anti-TAB1 antibody at either 1-cell or 2-cell stages, the immunoprecipates were detected by either anti-TAB1 antibody or anti-ubiquitin antibody. The results show that more Ub chains on TAB1 were detected in the two cell stage embryos than in the zygotes, when the TAB1 proteins were at comparable level.


( D ) RNF114 - dependent TAB1 degradation during early development . ( a - c ) Representive time lapse were shown for each treatment . Scale bar = 20um . ( a ' - c ' ) of GFP signals for respective treatments in ( a - c ) . n = 14 , 9 or 12 for NC , Rnf114 siRNA1 # or Rnf114 siRNA2 # treatment group respectively . Gray traces : Tab1 - GFP signals were normalized to first time point ; Black trace : average signal of for each treatment .

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(D) RNF114-dependent TAB1 degradation during early embryo development. (a-c) Representive time lapse embryo images were shown for each treatment. Scale bar = 20um. (a'-c') Quantification of the GFP fluorescent signals for respective treatments in (a-c). n=14, 9 or 12 for the NC, Rnf114 siRNA1# or Rnf114 siRNA2# treatment group respectively. Gray traces: Tab1-GFP fluorescent signals were normalized to the first time point; Black trace: average signal of all embryos for each treatment.


( E ) Tab1 knockdown partly rescued early development defect observed with Rnf114 knockdown . Both Rnf114 Tab1 siRNAs were injected into GV oocytes , then oocytes matured , were fertilized by IVF , fertilized eggs were transferred into KSOM media , cultured for 48 - 50 h . percentages of arrested at 2 - stage were counted . Three independent experiment replicates were performed , error bars represent s . d . ; * * : P < 0 . 01 in unpaired two - t - .

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(E) Tab1 knockdown partly rescued the early embryo development defect observed with Rnf114 knockdown. Both Rnf114 and Tab1 siRNAs were injected into the GV oocytes, then the oocytes matured, and were fertilized by IVF, and the fertilized eggs were transferred into KSOM media, cultured for 48-50 h. The percentages of embryos arrested at 2-cell stage were counted. Three independent experiment replicates were performed, error bars represent s.d.; **:P<0.01 in unpaired two-tailed t-test.


( A ) NF - κB activation during MZT . Immunofluorescence of p65 shows translocation of p65 at two - stage thereafter . Scale bar = 25um .

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(A) NF-κB activation during MZT. Immunofluorescence staining of p65 shows nuclear translocation of p65 at the two-cell stage and thereafter. Scale bar = 25um.


( B ) of IκBα its phosphorylated form ( p - IκBα ) during mouse early embryonic development were detected by western blotting , representative western blotting of IκBα p - IκBα was shown on left quantitation ( right panel ) indicated that of p - IκBα / total IκBα was gradually increased from MII oocytes to 4 - stage ( three independent experiment replicates were performed , error bars represent s . e . m . ) .

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(B) The protein levels of IκBα and its phosphorylated form (p-IκBα) during mouse early embryonic development were detected by western blotting, representative western blotting of IκBα and p-IκBα was shown on the left and the quantitation analysis (right panel) indicated that the ratio of p-IκBα/total IκBα was gradually increased from MII oocytes to 4-cell stage embryos (three independent experiment replicates were performed, error bars represent s.e.m.).


( C ) NF - κB pathway inhibition using specific inhibitor Bay11 - 7082 resulted in two stage arrest . Three independent experiment replicates were performed , error bars represent s . d . ; * * * : P < 0 . 001 in unpaired two - t - .

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(C) NF-κB pathway inhibition using the specific inhibitor Bay11-7082 resulted in two cell stage arrest. Three independent experiment replicates were performed, error bars represent s.d.; ***:P<0.001 in unpaired two-tailed t-test.


( D , E ) Representive immunofluorescence of p65 after knockdown of Rnf114 by Rnf114 siRNA # 1 or siRNA # 2 or overexpression of Tab1 by Tab1 mRNA injection ( shown in D . Scale bar = 25um . ) , percentages of two - stage with p65 translocation ( shown in E . ) were significantly decreased after Rnf114 knockdown or Tab1 overexpression ( chi - square was performed for statistical ) .

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(D, E) Representive immunofluorescence staining of p65 after knockdown of Rnf114 by Rnf114 siRNA #1 or siRNA #2 or overexpression of Tab1 by Tab1 mRNA injection (shown in D. Scale bar = 25um.), the percentages of two-cell stage embryos with p65 nuclear translocation (shown in E.) were significantly decreased after Rnf114 knockdown or Tab1 overexpression (chi-square test was performed for statistical analysis).


( F ) Western blot at 2 - stage showed of p - IκBα were downregulated in Rnf114 siRNA # 1 , siRNA # 2 injected groups .

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(F) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the Rnf114 siRNA #1, siRNA #2 injected groups.


( G ) Western blot at 2 - stage showed of p - IκBα were downregulated in exogenous Tab1 mRNA injected groups , ddH2O was injected as control . Dotted indicates a cropped lane .

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(G) Western blot analysis at 2-cell stage showed the levels of p-IκBα were downregulated in the exogenous Tab1 mRNA injected groups, ddH2O was injected as control. Dotted line indicates a cropped lane.


( A ) Confocal observation of mitochondrial potential after TMRM of LMP1 - negative or - positive NPC . Red , which indicates normal mitochondrial potential , was converted into green after a reduction in mitochondrial potential . were analyzed using Image J software ( scale bar , 10 μm ) . Data are presented as means ± S . E . M . ( paired t - , n = 6 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) .

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(A) Confocal observation of mitochondrial potential after TMRM staining of LMP1-negative or -positive NPC cells. Red fluorescence, which indicates normal mitochondrial potential, was converted into green fluorescence after a reduction in mitochondrial potential. Images were analyzed using Image J software (scale bar, 10 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01).


( C ) Extent of Ca2 + - mediated mitochondrial swelling in NPC . Data are presented as means ± S . E . M . ( paired t - , n = 6 , biological replicates per group , * * p < 0 . 01 ) .

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(C) Extent of Ca2+-mediated mitochondrial swelling in NPC cells. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, ** p < 0.01).


( A ) LMP1 is localized to inner mitochondrial . Isolated mitochondria were incubated with proteinase K ( 40 μM ) for 30 min before LMP1 expression was detected ( left ) ; mitochondria were further subjected to submitochondrial fractionation . VDAC1 , TIM23 , HSP60 were used to represent MOM , MIM , mitochondrial matrix , respectively ( right ) .

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(A) LMP1 is localized to the inner mitochondrial membrane. Isolated mitochondria were incubated with proteinase K (40 μM) for 30 min before LMP1 expression was detected (left); the mitochondria were further subjected to submitochondrial fractionation. VDAC1, TIM23, and HSP60 were used to represent MOM, MIM, and mitochondrial matrix protein, respectively (right).


( B ) interaction of LMP1 endogenous ANT1 was analyzed by an immunofluorescence confocal . HK1 - LMP1 were immunostained with anti - LMP1 ( green ) anti - ANT1 ( red ) antibodies then subjected to confocal microscopy ， use imageJ to calculate co - location level of both . nuclei were stained with DAPI ( scale bar , 5 μm ) . quantified graph on top right shows of yellow ( merge ) to red ( ANT1 ) in HK1 - LMP1 . Data are presented as means ± S . E . M . ( n = 6 , biological replicates per group ) .

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(B) The interaction of LMP1 and endogenous ANT1 was analyzed by an immunofluorescence confocal assay. HK1-LMP1 cells were immunostained with anti-LMP1 (green) and anti-ANT1 (red) antibodies and then subjected to confocal microscopy，and use imageJ to calculate the co-location level of both. The nuclei were stained with DAPI (scale bar, 5 μm). The quantified graph on the top right shows the percentage of yellow fluorescence (merge) to red fluorescence (ANT1) in HK1-LMP1 cells. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).


( G ) LMP1 binds directly to ANT1 dependent on its transmembrane domain in 293T . IP / IB was used to detect interaction between Flag - tagged LMP1 mutants Myc - tagged ANT1 . IP of lysates using a Flag or Myc antibody .

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(G) LMP1 binds directly to ANT1 dependent on its transmembrane domain in 293T cells. IP/IB was used to detect the interaction between Flag-tagged LMP1 mutants and Myc-tagged ANT1. IP of cell lysates using a Flag or Myc antibody.


( A Effect of inhibitors BKA CATR on ANT1 - VDAC1 binding in 293T .

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(A Effect of inhibitors BKA and CATR on ANT1-VDAC1 binding in 293T cells.


B ) Effect of inhibitors BKA CATR on ANT1 - VDAC1 binding in 293T . Data are presented as means ± S . E . M . ( paired t - , n = 3 , biological replicates per group , * * p < 0 . 01 ) .

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B) Effect of inhibitors BKA and CATR on ANT1-VDAC1 binding in 293T cells. Data are presented as means ± S.E.M. (paired t-test, n = 3, biological replicates per group, ** p < 0.01).


( C effect of different amounts of EBV - LMP1 on binding of ANT1 - VDAC1 was detected by CO - IP .

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(C The effect of different amounts of EBV-LMP1 on the binding of ANT1-VDAC1 was detected by CO-IP.


D ) effect of different amounts of EBV - LMP1 on binding of ANT1 - VDAC1 was detected by CO - IP . Data are presented as means ± S . E . M . ( paired t - , n = 3 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) .

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D) The effect of different amounts of EBV-LMP1 on the binding of ANT1-VDAC1 was detected by CO-IP. Data are presented as means ± S.E.M. (paired t-test, n = 3, biological replicates per group, * p < 0.05, ** p < 0.01).


( G ) 19F - NMR spectra of ANT1 - Y195 - F2Y ANT1 - Y290 - F2Y after titration with different amounts of LMP1 . After incubation at room for 30 min , nuclei were continuously monitored at 500 MZ for 30 min . ( H ) 19F - NMR spectra of ANT1 - Y195 - F2Y ANT1 - Y290 - F2Y after titration with different amounts of LMP1 - ΔT . After incubation at room for 30 min , nuclei were continuously monitored at 500 MZ for 30 min .

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(G) 19F-NMR spectra of ANT1-Y195-F2Y and ANT1-Y290-F2Y after titration with different amounts of LMP1. After incubation at room temperature for 30 min, the nuclei were continuously monitored at 500 MZ for 30 min. (H) 19F-NMR spectra of ANT1-Y195-F2Y and ANT1-Y290-F2Y after titration with different amounts of LMP1-ΔT. After incubation at room temperature for 30 min, the nuclei were continuously monitored at 500 MZ for 30 min.


( A - B ) ADP / ATP exchange rate in EBV - LMP1 - positive nasopharyngeal carcinoma is decreased . value of Magnesium Green ™ ( 1 μM ) was measured at 505 nm ( Ex ) / 535 nm ( Em ) by enzymography ( A ) , ADP / ATP exchange rate was calculated after conversion to ATP ( B ) . Data are presented as means ± S . E . M . ( paired t - , n = 5 , biological replicates per group , * * p < 0 . 01 ) .

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(A-B) The ADP/ATP exchange rate in EBV-LMP1-positive nasopharyngeal carcinoma cells is decreased. The fluorescence value of Magnesium Green ™ (1 μM) was measured at 505 nm (Ex)/535 nm (Em) by enzymography (A), and the ADP/ATP exchange rate was calculated after conversion to ATP (B). Data are presented as means ± S.E.M. (paired t-test, n = 5, biological replicates per group, ** p < 0.01).


( C ) LMP1 - negative or - positive NPC were treated with BKA ( 1 μM ) or CATR ( 5 μM ) for 24 h , Mitochondrial OCR assessed with XF24 extracellular flux analyzer ( Seahorse ) under sequential treatment with oligomycin , FCCP , antimycin A . Data are presented as means ± S . E . M . ( n = 6 , biological replicates per group ) . ( D ) Oxidative phosphorylation - related metrics on OCR datas . Data are presented as means ± S . E . M . ( paired t - , n = 6 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) .

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(C) LMP1-negative or -positive NPC cells were treated with BKA (1 μM) or CATR (5 μM) for 24 h, Mitochondrial OCR assessed with the XF24 extracellular flux analyzer (Seahorse) under sequential treatment with oligomycin, FCCP, and antimycin A. Data are presented as means ± S.E.M. (n = 6, biological replicates per group). (D) Oxidative phosphorylation-related metrics based on OCR datas. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05,** p < 0.01).


( A ) HK1 - LMP1 were treated with increasing concentrations of cisplatin for 24 h . viability was determined by MTS . Data are presented as means ± S . E . M . ( n = 6 , biological replicates per group ) .

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(A) HK1-LMP1 were treated with increasing concentrations of cisplatin for 24 h. Cell viability was determined by MTS assay. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).


( D ) IC50 value of combination of CATR cisplatin in CNE1 - LMP1 was changed . Data are presented as means ± S . E . M . ( n = 6 , biological replicates per group ) .

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(D) The IC50 value of the combination of CATR and cisplatin in CNE1-LMP1 cells was changed. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).


( G ) Flow cytometry of of NPC treated for 24 h with CATR ( 1 μM ) alone , cisplatin ( 20 μM ) alone , or cisplatin ( 20 μM ) combined with CATR ( 1 μM ) . Data are presented as means ± S . E . M . ( paired t - , n = 5 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) .

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(G) Flow cytometry analysis of death of NPC cells treated for 24 h with CATR (1 μM) alone, cisplatin (20 μM) alone, or cisplatin (20 μM) combined with CATR (1 μM). Data are presented as means ± S.E.M. (paired t-test, n = 5, biological replicates per group, * p < 0.05, ** p < 0.01).


( H ) Trypan blue was used to detect of NPC treated for 24 h with vehicle , cisplatin , CATR , or a combination of cisplatin CATR ( 1 μM ) . Data are presented as means ± S . E . M . ( paired t - , n = 6 , biological replicates per group , * * p < 0 . 01 ) .

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(H) Trypan blue staining was used to detect death of NPC cells treated for 24 h with vehicle, cisplatin, CATR, or a combination of cisplatin and CATR (1 μM). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, ** p < 0.01).


( F ) Immunohistochemistry examination of of Ki67 ( scale bar , 50 μm ) . Data are presented as means ± S . E . M . ( paired t - , n = 6 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) .

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(F) Immunohistochemistry examination of the protein levels of Ki67 (scale bar, 50 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01).


( G ) Apoptosis was analyzed by TUNEL ( scale bar , 50 μm ) . Data are presented as means ± S . E . M . ( paired t - , n = 6 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) . ( H ) Apoptosis was analyzed by Western blotting .

(G) Apoptosis was analyzed by TUNEL assay (scale bar, 50 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01). (H) Apoptosis was analyzed by Western blotting.


( A ) Michaelis - Menten of reduction of 2 - hydroxyacetophenone ( 2 - HAP ) . initial velocities ( V0 / [ Etot ] ) are given for subunits ( i . e . , normalized to subunit not oligomer ) . Enzyme assays were conducted at 30 ˚ C in 100 mM sodium phosphate ( pH 6 . 0 ) using 500 µM NADPH 100 nM SCR or 1 µM SCR - his6 . Enzyme activity at each substrate was assayed at least three usually four times . data points show average values for these technical replicates , while error bars show standard deviations .

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(A) Michaelis-Menten analysis of the reduction of 2-hydroxyacetophenone (2-HAP). The initial velocities (V0/[Etot]) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH and 100 nM SCR or 1 µM SCR-his6. Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.


Comparison of kcat values obtained from Michaelis - Menten analyses of reduction of four different substrates . turnover rates ( kcat ) are given for subunits ( i . e . , normalized to subunit not oligomer ) . Enzyme assays were conducted at 30 ˚ C in 100 mM sodium phosphate ( pH 6 . 0 ) using 500 µM NADPH 100 nM SCR or 1 µM SCR - his6 . data points show average values for these technical replicates , while error bars show standard deviations .

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Comparison of kcat values obtained from Michaelis-Menten analyses of the reduction of four different substrates. The turnover rates (kcat) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH and 100 nM SCR or 1 µM SCR-his6. The data points show the average values for these technical replicates, while the error bars show the standard deviations.


( A ) Analytical gel - filtration of purified his8 - MBP - SCR2 ( black ) , strep - SCR ( yellow ) , his8 - MBP - SCR2 / strep - SCR on a Superdex 200 Increase column at 20 ˚ C in 150 mM NaCl , 50 mM Tris - HCl , pH 8 . 0 .

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(A) Analytical gel-filtration of purified his8-MBP-SCR2 (black), strep-SCR (yellow), and his8-MBP-SCR2/strep-SCR on a Superdex 200 Increase column at 20 ˚C in 150 mM NaCl, 50 mM Tris-HCl, pH 8.0.


( B ) Michaelis - Menten kinetic analyses of NADPH - dependent reduction of 2 - hydroxyacetophenone methyl acetoacetate at 30 ˚ C in 100 mM sodium phosphate , pH 6 . 0 . turnover rate ( V0 / [ Etot ] ) is given for subunits Enzyme activity at each substrate was assayed at least three usually four times . data points show average values for these technical replicates , while error bars show standard deviations .

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(B) Michaelis-Menten kinetic analyses of NADPH-dependent reduction of 2-hydroxyacetophenone and methyl acetoacetate at 30 ˚C in 100 mM sodium phosphate, pH 6.0. The turnover rate (V0/[Etot]) is given for the individual subunits Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.


Western blotting for mTORC1 mTORC2 activity in mESCs transduced with Rptor Rictor shRNAs .

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Western blotting for mTORC1 and mTORC2 activity in mESCs transduced with Rptor and Rictor shRNAs.


of mESCs transduced with Rptor Rictor shRNAs in 2iL . Scale bar , 400 µm . Relative proliferation of mESCs as in ( E ) ( mean values ± SD , P value was calculated using two - unpaired Student ' s t , * P < 0 . 05 , also hereafter in similar experiments ) . n = 3 biological replicates .

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Images of mESCs transduced with Rptor and Rictor shRNAs in 2iL. Scale bar, 400 µm.   Relative proliferation of mESCs as in (E) (mean values ± SD, P value was calculated using two-tailed unpaired Student's t test, *P < 0.05, also hereafter in similar experiments). n = 3 biological replicates.


Western blotting for RPTOR indicated pluripotency in mESCs as in ( H ) ( left ) , of pluripotency ( right ) ( mean values ± SD ) . n = 3 biological replicates .

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Western blotting for RPTOR and the indicated pluripotency proteins in mESCs as in (H) (left), and quantification of pluripotency proteins (right) (mean values ± SD). n = 3 biological replicates.


of mESCs transduced with Rptor siRNA on different days in 2iL . siNC , transduced with a negative control ; day 0 was day of siRNA transfection , also hereafter in similar experiments . Scale bar , 400 µm .

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Images of mESCs transduced with Rptor siRNA on different days in 2iL. siNC, transduced with a negative control; day 0 was the day of siRNA transfection, also hereafter in similar experiments. Scale bar, 400 µm.


- seq heatmaps of indicated pluripotency gene sets in control INK128 ( 200 nM ) treated mESCs in 2iL . - seq heatmaps of indicated pluripotency gene sets in Rptor WT KO mESCs in 2iL .

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RNA-seq heatmaps of the indicated pluripotency gene sets in control and INK128 (200 nM) treated mESCs in 2iL.   RNA-seq heatmaps of indicated pluripotency gene sets in Rptor WT and KO mESCs in 2iL.


of mESCs transduced with Eif4a1 , Eif4e Eif4g1 shRNAs in 2iL . Scale bar , 400 µm . Relative proliferation of mESCs as in ( B ) ( mean values ± SD ) . n = 3 biological replicates .

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Images of mESCs transduced with Eif4a1, Eif4e and Eif4g1 shRNAs in 2iL. Scale bar, 400 µm.   Relative proliferation of mESCs as in (B) (mean values ± SD). n = 3 biological replicates.


Western blotting for indicated shRNAs pluripotency as in ( B ) ( left ) ( right ) ( mean values ± SD ) . n = 3 biological replicates .

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Western blotting for the indicated shRNAs and pluripotency proteins as in (B) (left) and quantification (right) (mean values ± SD). n =3 biological replicates.


Relative proliferation of mESCs as in ( B ) ( mean values ± SD , P value was calculated using two - unpaired Student ' s t , * * P < 0 . 01 , also hereafter in similar experiments ) . n = 3 biological replicates .

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Relative proliferation of mESCs as in (B) (mean values ± SD, P value was calculated using two-tailed unpaired Student's t test, **P < 0.01, also hereafter in similar experiments). n = 3 biological replicates.


of mESCs transduced with Larp1 shRNAs in 2iL , in control or INK128 ( 200 nM ) treated for three days . Scale bar , 400 µm . Relative proliferation of mESCs as in ( E ) ( mean values ± SD ) . n = 3 biological replicates .

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Images of mESCs transduced with Larp1 shRNAs in 2iL, in control or INK128 (200 nM) treated for three days. Scale bar, 400 µm.   Relative proliferation of mESCs as in (E) (mean values ± SD). n = 3 biological replicates.


Relative enrichment heatmaps of mitochondrial ribosome subunit ETC component mRNAs as in ( A ) .

Relative enrichment heatmaps of mitochondrial ribosome subunit and ETC complex component mRNAs as in (A).


RT - qPCR for pluripotency mitochondrial ribosome subunit genes as in ( E ) ( mean values ± SD ) . n = 3 biological replicates .

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RT-qPCR for pluripotency and mitochondrial ribosome subunit genes as in (E) (mean values ± SD). n = 3 biological replicates.


OCR of INK128 ( 200 nM ) treated mESCs in 2iL .

OCR analysis of INK128 (200 nM) treated mESCs in 2iL.


qPCR for mtDNA copy number in mESCs treated with INK128 or transduced with Rptor shRNAs in 2iL ( mean values ± SD ) . n = 3 biological replicates .

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qPCR for mtDNA copy number in mESCs treated with INK128 or transduced with Rptor shRNAs in 2iL (mean values ± SD). n = 3 biological replicates.


Western blotting for indicated pluripotency in mESCs as in ( F ) ( left ) , ( right ) ( mean values ± SD ) . n = 3 biological replicates .

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Western blotting for the indicated pluripotency proteins in mESCs as in (F) (left), and quantification (right) (mean values ± SD). n = 3 biological replicates.


of mESCs transduced with Rptor or Rictor shRNAs in SL . Scale bar , 400 µm . Relative proliferation of mESCs as in ( A ) ( mean values ± SD ) . n = 3 biological replicates .

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Images of mESCs transduced with Rptor or Rictor shRNAs in SL. Scale bar, 400 µm.   Relative proliferation of mESCs as in (A) (mean values ± SD). n = 3 biological replicates.


of mESCs transduced with Eif4a1 , Eif4e Eif4g1 shRNAs in SL . Scale bar , 400 µm .

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Images of mESCs transduced with Eif4a1, Eif4e and Eif4g1 shRNAs in SL. Scale bar, 400 µm.


Western blotting for EIF4E , EIF4G1 indicated pluripotency in with indicated KD in indicated culture conditions .

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Western blotting for EIF4E, EIF4G1 and the indicated pluripotency proteins in cells with the indicated KD in the indicated culture conditions.


Immunoblot comparing UFL1 autoUFMylation ( Top ) UFM1 chain synthesis ( Middle ) in presence of UFL1 expressed alone in with UFBP1 . ( Bottom ) Assays to monitor UFMylation of substrates using purified TRIP4 . Reaction products were separated on a 4 - 12 % SDS - PAGE gel under reducing conditions analysed by immunoblotting using indicated antibodies .

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Immunoblot comparing UFL1 autoUFMylation (Top) and UFM1 chain synthesis (Middle) in the presence of UFL1 expressed alone and in complex with UFBP1. (Bottom) Assays to monitor UFMylation of substrates using purified TRIP4. Reaction products were separated on a 4-12% SDS-PAGE gel under reducing conditions and analysed by immunoblotting using indicated antibodies.


In vitro reconstitution of 60S ribosome UFMylation . Purified 60S ribosome was incubated with either UFL1 on its own or UFL1 / UFBP1 together with UBA5 , UFC1 UFM1 for indicated time points at 30 ° C .

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In vitro reconstitution of 60S ribosome UFMylation. Purified 60S ribosome was incubated with either UFL1 on its own or UFL1/UFBP1 complex together with UBA5, UFC1 and UFM1 for indicated time points at 30°C.


Immunoblot showing formation of free UFM1 chains in presence ( Lane 3 , 4 ) absence of UFL1 / UFBP1 ( Lane 1 , 2 ) . ( Bottom ) Graphical representation of linkage composition of di - UFM1 chains obtained from LC - MS / MS .

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Immunoblot showing formation of free UFM1 chains in the presence (Lane 3,4) and absence of UFL1/UFBP1 (Lane 1,2). (Bottom) Graphical representation of the linkage composition of di-UFM1 chains obtained from LC-MS/MS analysis.


Immunoblot analysing polyUFMylation ( top ) UFL1 autoUFMylation ( bottom ) in presence of indicated Lys to Arg ( K - R ) Lys - less ( K0 ) mutants of UFM1 .

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Immunoblot analysing polyUFMylation (top) and UFL1 autoUFMylation (bottom) in the presence of indicated Lys to Arg (K-R) and Lys-less(K0) mutants of UFM1.


Immunoblot showing in vitro UFMylation to check for formation of di - UFM1 chains in presence of WT , Lys - less ( K0 ) single lys mutants ( K only ) of UFM1 .

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Immunoblot showing in vitro UFMylation assay to check for formation of di-UFM1 chains in the presence of WT, Lys-less (K0) and single lys mutants (K only) of UFM1.


Coomassie stained gels monitoring discharge of UFM1 from UFC1 in presence of indicated free . UBE2D3 is used as a positive control for lysine cysteine discharge .

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Coomassie stained gels monitoring discharge of UFM1 from UFC1 in the presence of indicated free amino acids. UBE2D3 is used as a positive control for lysine and cysteine discharge.


Immunoblot showing in vitro UFMylation assays in presence of Cys to Ala ( indicated as C - A ) mutants of UFL1 to check for UFBP1 UFMylation ( top ) , UFL1 autoUFMylation ( middle ) substrate UFMylation ( bottom ) .

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Immunoblot showing in vitro UFMylation assays in the presence of Cys to Ala (indicated as C-A) mutants of UFL1 to check for UFBP1 UFMylation (top), UFL1 autoUFMylation (middle) and substrate UFMylation (bottom).


Single - turnover lysine discharge assays in presence absence of full UFL1 / UFBP1 . reaction was stopped by addition of non - reducing SDS loading buffer ( 1x final ) analysed for discharge on a 4 - 12 % SDS PAGE gel under non - reducing conditions . Top gel - LI - COR scan of fluorescently labelled UFM1 ( UFM1 * ) ; bottom gel - Coomassie

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Single-turnover lysine discharge assays in the presence and absence of full length UFL1/UFBP1 complex. The reaction was stopped by addition of non-reducing SDS loading buffer (1x final) and analysed for discharge on a 4-12% SDS PAGE gel under non-reducing conditions. Top gel - LI-COR scan of fluorescently labelled UFM1(UFM1*); bottom gel - Coomassie stained


Immunoblot from immunoprecipitation ( IP ) assays to validate predicted model of UFL1 / UFBP1 interaction in . UFL1WT - 3xFLAG UFL1L45R - 3xFLAG were transiently overexpressed along with UFBP1 - SBP in HEK293T - UFL1 KO subjected to separate pulldowns using anti - FLAG antibody or streptavidin . Immunoprecipitated material was run on a 4 - 12 % SDS PAGE gel analysed by immunoblotting using indicated antibodies .

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Immunoblot from immunoprecipitation (IP) assays to validate the predicted model of UFL1/UFBP1 interaction in cells. UFL1WT-3xFLAG and UFL1L45R-3xFLAG were transiently overexpressed along with UFBP1-SBP in HEK293T-UFL1 KO cells and subjected to separate pulldowns using anti-FLAG antibody or streptavidin. Immunoprecipitated material was run on a 4-12% SDS PAGE gel and analysed by immunoblotting using indicated antibodies.


Immunoblot showing in vitro UFMylation to monitor formation of free di - UFM1 chains in presence of UFL1 / UFBP1 complexes bearing different domain boundaries .

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Immunoblot showing in vitro UFMylation assay to monitor formation of free di-UFM1 chains in the presence of UFL1/UFBP1 complexes bearing different domain boundaries.


Immunoblot of in vitro monitoring UFMylation of 60S Ribosome in presence of different UFL1 / UFBP1 truncations as depicted in ( E ) .

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Immunoblot of in vitro assay monitoring UFMylation of 60S Ribosome in the presence of different UFL1/UFBP1 truncations as depicted in (E).


In vitro UFMylation in presence of increasing concentrations of CDK5RAP3 to monitor E3 ligase activity of UFL1 / UFBP1 . reaction products were run on a 4 - 12 % SDS - PAGE gel under reducing conditions immunoblotting was performed using indicated antibodies .

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In vitro UFMylation assay in the presence of increasing concentrations of CDK5RAP3 to monitor the E3 ligase activity of UFL1/UFBP1 complex. The reaction products were run on a 4-12% SDS-PAGE gel under reducing conditions and immunoblotting was performed using indicated antibodies.


Single turnover lysine discharge assays to check for activation of UFC1 by UFL1 / UFBP1 in presence absence of CDK5RAP3 . reaction products were run on a 4 - 12 % SDS PAGE visualized by Oriole . of discharge of UFM1 from UFC1 in presence absence of CDK5RAP3 as seen in Fig 4D , n = 3 biological replicates ,

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Single turnover lysine discharge assays to check for activation of UFC1 by UFL1/UFBP1 in the presence and absence of CDK5RAP3. The reaction products were run on a 4-12% SDS PAGE and visualized by Oriole staining.   Quantification of discharge of UFM1 from UFC1 in the presence and absence of CDK5RAP3 as seen in Fig 4D, n=3 biological replicates,


Substrate UFMylation assays using purified 60S Ribosomes in presence of increasing of CDK5RAP3 . Reaction was performed for 10 min , stopped by addition of SDS Loading dye analysed on an SDS - PAGE gel under reducing conditions followed by immunoblotting with indicated antibodies .

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Substrate UFMylation assays using purified 60S Ribosomes in the presence of increasing concentration of CDK5RAP3. Reaction was performed for 10 min, stopped by addition of SDS Loading dye and analysed on an SDS-PAGE gel under reducing conditions followed by immunoblotting with indicated antibodies.


Immunoblot monitoring UFMylation of Histone H4 in presence of increasing concentrations of CDK5RAP3 ( 1 µM , 2 µM , 3 µM ) . UFL1 / UFBP1 concentrations - 1 µM .

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Immunoblot analysis monitoring UFMylation of Histone H4 in the presence of increasing concentrations of CDK5RAP3 (1 µM, 2 µM, 3 µM). UFL1/UFBP1 concentrations - 1 µM.


Immunoblot showing in vitro UFMylation to compare activity of UFC1 Wild type mutants in presence of UFL1 / UFBP1 . In vitro UFMylation to compare activity of UFC1 Wild type UFC1 T106I in presence of UFL1 / UFBP1 .

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Immunoblot showing the in vitro UFMylation assay to compare the activity of UFC1 Wild type and mutants in the presence of UFL1/UFBP1 complex.   In vitro UFMylation assay to compare the activity of UFC1 Wild type and UFC1 T106I in the presence of UFL1/UFBP1 complex.


Substrate UFMylation assays using purified 60S Ribosomes in presence of UFC1 Wild type different mutants of UFC1 .

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Substrate UFMylation assays using purified 60S Ribosomes in the presence of UFC1 Wild type and different mutants of UFC1.


In vitro UFMylation assays in presence of Wild type UFC1 ( UFC1WT ) UFC1 lacking N - terminal helix ( UFC1ΔN ) .

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In vitro UFMylation assays in the presence of Wild type UFC1 (UFC1WT) and UFC1 lacking the N-terminal helix (UFC1ΔN).


to compare Lys discharge activities of UFC1WT UFC1ΔN on its own , in presence of UFL1 / UFBP1 or in presence of preassembled UFL1 / UFBP1 / CDK5RAP3 . Top gel : LI - COR scan of fluorescently labelled UFM1 ( UFM1 * ) ; bottom gel - Coomassie stained ( Representative of three independent experiments ) .

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Assay to compare Lys discharge activities of UFC1WT and UFC1ΔN on its own, in the presence of UFL1/UFBP1 or in the presence of preassembled UFL1/UFBP1/CDK5RAP3 complex. Top gel: LI-COR scan of fluorescently labelled UFM1(UFM1*); bottom gel - Coomassie stained (Representative of three independent experiments).


H . Effect of deletion of N - term helix of UFC1 in 60S ribosome UFMylation . Purified 60S ribosomes ( 50 nM ) were incubated with 0 . 5 µM UBA5 , 1 µM UFM1 , 100 nM UFL1 / UFBP1 in presence of either 1 µM UFC1WT or 1 µM UFC1ΔN at 30 ° C . reaction was stopped at indicated time points run on a 4 - 12 % SDS PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies . ( Right - shorter time duration . Left - Longer time duration ) .

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H. Effect of deletion of N -term helix of UFC1 in 60S ribosome UFMylation. Purified 60S ribosomes (50 nM) were incubated with 0.5 µM UBA5, 1 µM UFM1, 100 nM UFL1/UFBP1 complex in the presence of either 1 µM UFC1WT or 1 µM UFC1ΔN at 30°C. The reaction was stopped at indicated time points and run on a 4-12% SDS PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies. (Right - shorter time duration. Left - Longer time duration).


B HeLa expressing GFP - parkin ( green ) , OCT - DsRed2 ( red ) CFP - Drp1K38E were left untreated ( untreated ) or treated for 2 h with 10 µM CCCP ( CCCP ) , then fixed stained for TOM20 ( blue ) . Scale bars , 30 µm .

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B HeLa cells expressing GFP-parkin (green), OCT-DsRed2 (red) and CFP-Drp1K38E were left untreated (untreated) or treated for 2 h with 10 µM CCCP (CCCP), then fixed and stained for TOM20 (blue). Scale bars, 30 µm.


C prepared as in ( B ) were treated with 50 µM antimycin A for 2 h . Arrows indicate OCT - DsRed2 - positive / TOM20 - negative MDVs that colocalize with GFP - parkin , while circles indicate MDVs that are parkin - negative . Arrowheads indicate nascent vesicles , adjacent to mitochondria , exhibiting cargo selectivity parkin recruitment . Open arrowheads indicate parkin - negative MDVs containing reciprocal cargo ( TOM20 - positive / OCT - DsRed2 - negative ) . boundaries are delineated in GFP - parkin single - channel image . Scale bars , 30 µm .

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C Cells prepared as in (B) were treated with 50 µM antimycin A for 2 h. Arrows indicate OCT-DsRed2-positive/TOM20-negative MDVs that colocalize with GFP-parkin, while circles indicate MDVs that are parkin-negative. Arrowheads indicate nascent vesicles, adjacent to mitochondria, exhibiting cargo selectivity and parkin recruitment. Open arrowheads indicate parkin-negative MDVs containing the reciprocal cargo (TOM20-positive/OCT-DsRed2-negative). Cell boundaries are delineated in the GFP-parkin single-channel image. Scale bars, 30 µm.


A of number of OCTμDsRed2μpositive / TOM20μnegative vesicles in expressing GFP , GFPμparkinWT , GFPμparkinR42P , GFPμparkinK211N , or GFPμparkinC431F , treated with or without antimycin A ( anti A ) as in ( C ) ; both total number ( bars ) number colocalizing with GFPμparkin ( gray bars ) are indicated . Bars represent mean ± s . e . m . Pμvalues are given first for GFPμ / GFPμparkinμpositive vesicles , then for total vesicle number ( n = 49 - 68 in 2 - 3 experiments ) ; ns , not significant ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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A Quantification of the number of OCTμDsRed2μpositive/TOM20μnegative vesicles in cells expressing GFP, GFPμparkinWT, GFPμparkinR42P, GFPμparkinK211N, or GFPμparkinC431F, treated with or without antimycin A (anti A) as in (C); both the total number (white bars) and the number colocalizing with GFPμparkin (gray bars) are indicated. Bars represent the mean ± s.e.m. Pμvalues are given first for GFPμ/GFPμparkinμpositive vesicles, then for total vesicle number (n = 49-68 cells in 2-3 experiments); ns, not significant; **P < 0.01; ***P < 0.001).


C HeLa expressing various GFP - parkin mutant contructs ( green ) , pOCT - DsRed2 ( red ) CFP - Drp1K38E were treated with 50 µM antimycin A for 2 h , then fixed immunostained against TOM20 . Arrows indicate matrix - positive / TOM20 - negative structures colocalizing with GFP - parkin . Scale bars , 30 µm .

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C HeLa cells expressing various GFP-parkin mutant contructs (green), pOCT-DsRed2 (red) and CFP-Drp1K38E were treated with 50 µM antimycin A for 2 h, then fixed and immunostained against TOM20. Arrows indicate matrix-positive/TOM20-negative structures colocalizing with GFP-parkin. Scale bars, 30 µm.


D Immunoblot of whole - lysates of HeLa expressing various GFP - parkin constructs , treated with 50 µM antimycin A ( anti A , + ) or DMSO ( - ) for 2 h .

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D Immunoblot of whole-cell lysates of HeLa cells expressing various GFP-parkin constructs, treated with 50 µM antimycin A (anti A, +) or DMSO (-) for 2 h.


A Representative immunoblot of whole ‐ lysates from U2OS : GFP U2OS : GFP ‐ parkin transfected with non ‐ targeting siRNA or siRNA targeting Drp1 ( siDrp1 ) .

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A Representative immunoblot of whole‐cell lysates from U2OS:GFP and U2OS:GFP‐parkin cells transfected with non‐targeting siRNA or siRNA targeting Drp1 (siDrp1).


B U2OS : GFP − parkin transfected with siRNA targeting Drp1 were treated with DMSO ( upper panels ) or 25 μM antimycin A ( anti A , lower panels ) for 90 min , then fixed immunostained against PDH ( red ) TOM20 ( blue ) . PDHE2 / E3 bp − positive / TOM20 − negative MDVs colocalizing with GFP − parkin ( arrows ) or not ( circles ) are indicated . Scale bars , 20 μm ( first panels on left ) 2 μm .

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B U2OS:GFP−parkin cells transfected with siRNA targeting Drp1 were treated with DMSO (upper panels) or 25 μM antimycin A (anti A, lower panels) for 90 min, then fixed and immunostained against PDH (red) and TOM20 (blue). PDHE2/E3 bp−positive/TOM20−negative MDVs colocalizing with GFP−parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm (first panels on left) and 2 μm.


C of PDH − positive / TOM20 − negative vesicles in U2OS : GFP U2OS : GFP − parkin transfected with indicated siRNA , treated with DMSO or antimycin A ( anti A ) as in ( B ) ; both total number ( bars ) number colocalizing with GFP − parkin ( gray bars ) are indicated . Bars represent mean ± s . e . m . P − values are given first for GFP − / GFP − parkin − positive vesicles , then for total vesicle number ( n = 48 - 85 in 2 - 3 experiments ) ; ns , not significant ; * * * P < 0 . 001 ) .

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C Quantification of PDH−positive/TOM20−negative vesicles in U2OS:GFP and U2OS:GFP−parkin cells transfected with the indicated siRNA, treated with DMSO or antimycin A (anti A) as in (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 48-85 cells in 2-3 experiments); ns, not significant; ***P < 0.001).


D 5 μm ‐ long profile of parkin ‐ positive vesicle adjacent mitochondrial tubule depicted in antimycin A ‐ treated from ( B ) ( left ) . Increasing position values on x ‐ axis of plot ( right ) correspond to moving from top to bottom of profile . * MDV indicated by an arrow in ( B ) .

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D 5 μm‐long profile of the parkin‐positive vesicle and adjacent mitochondrial tubule depicted in the antimycin A‐treated cell from (B) (left). Increasing position values on the x‐axis of the fluorescence intensity plot (right) correspond to moving from the top to the bottom of the profile. *MDV indicated by an arrow in (B).


E of between centres of GFP − parkin − negative ( black bar ) or − positive ( gray bar ) vesicles edge of nearest mitochondrial tubule for vesicles quantified in antimycin A − treated U2OS : GFP − parkin transfected with siDrp1 in ( C ) . Error bars represent mean ± s . e . m . ; * P < 0 . 05 ( obtained by Student ' s t − ) .

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E Quantification of the distance between the centres of GFP−parkin−negative (black bar) or −positive (gray bar) vesicles and the edge of the nearest mitochondrial tubule for the vesicles quantified in antimycin A−treated U2OS:GFP−parkin cells transfected with siDrp1 in (C). Error bars represent the mean ± s.e.m.; *P < 0.05 (obtained by Student's t−test).


F U2OS : GFP ‐ parkin transfected with siRNA targeting Drp1 ( siDrp1 ) were treated with 25 μM antimycin A for 90 min prior to fixation . Samples were immunostained against TOM20 ( blue ) indicated mitochondrial marker ( red ) . TOM20 ‐ negative MDVs containing specified cargo colocalizing with GFP ‐ parkin ( arrows ) or not ( circles ) are indicated . Arrowheads show lack of colocalization between GFP ‐ parkin indicated mitochondrial marker . Scale bars , 20 μm ( first two columns of panels ) 2 μm ( remaining columns of panels ) .

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F U2OS:GFP‐parkin cells transfected with siRNA targeting Drp1 (siDrp1) were treated with 25 μM antimycin A for 90 min prior to fixation. Samples were immunostained against TOM20 (blue) and the indicated mitochondrial marker (red). TOM20‐negative MDVs containing the specified cargo and colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Arrowheads show lack of colocalization between GFP‐parkin and the indicated mitochondrial marker. Scale bars, 20 μm (first two columns of panels) and 2 μm (remaining columns of panels).


G of TOM20 − negative structures that stained positively for indicated cargo in U2OS : GFP ( GFP ) U2OS : GFP − parkin ( GFP − parkin ) , transfected with siRNA targeting Drp1 , treated with antimycin A ( anti A ) ; both total number ( bars ) number colocalizing with GFP − parkin ( gray bars ) are indicated . Error bars represent mean ± s . e . m . P − values are given first for GFP − / GFP − parkin − positive vesicles , then for total vesicle number ( n = 24 - 59 in two experiments ; ns , not significant ; * * * P < 0 . 001 ) .

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G Quantification of TOM20−negative structures that stained positively for the indicated cargo in U2OS:GFP (GFP) and U2OS:GFP−parkin (GFP−parkin) cells, transfected with siRNA targeting Drp1, treated with antimycin A (anti A); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 24-59 cells in two experiments; ns, not significant; ***P < 0.001).


AHeLa , transfected with siRNA targeting Drp1 GFP ‐ parkin ( green ) , were treated with 25 μM antimycin A for 90 min following a 30 ‐ min pretreatment with 50 nM bafilomycin A1 , then fixed immunostained against PDH E2 / E3 bp ( red ) TOM20 ( blue ) . PDH E2 / E3 bp ‐ positive / TOM20 ‐ negative MDVs colocalizing with GFP ‐ parkin ( arrows ) or not ( circles ) are indicated . Scale bar , 30 μm .

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AHeLa cells, transfected with siRNA targeting Drp1 and GFP‐parkin (green), were treated with 25 μM antimycin A for 90 min following a 30‐min pretreatment with 50 nM bafilomycin A1, then fixed and immunostained against PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp‐positive/TOM20‐negative MDVs colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Scale bar, 30 μm.


B of PDH E2 / E3bp ‐ positive / TOM20 ‐ negative vesicles in HeLa treated with antimycin A in presence of lysosomal inhibitors bafilomycin A1 or pepstatin A E ‐ 64d ; both total number ( bars ) number colocalizing with GFP ‐ parkin ( gray bars ) are indicated . Bars represent mean ± s . e . m . P ‐ values are given for total vesicle number ( n = 19 to 120 in 2 - 3 experiments ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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B Quantification of PDH E2/E3bp‐positive/TOM20‐negative vesicles in HeLa cells treated with antimycin A in the presence of the lysosomal inhibitors bafilomycin A1 or pepstatin A and E‐64d; both total number (white bars) and the number colocalizing with GFP‐parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P‐values are given for total vesicle number (n = 19 to 120 cells in 2-3 experiments; **P < 0.01; ***P < 0.001).


CHeLa , transfected with GFP ‐ parkin ( green ) siRNA targeting Drp1 Atg5 , were treated with 25 μM antimycin A for 90 min , then fixed , immunostained for PDH ( red ) TOM20 ( blue ) , counterstained for Hoescht ( gray ) . PDH E2 / E3bp bp ‐ positive / TOM20 ‐ negative MDVs colocalizing with GFP ‐ parkin ( arrows ) or not ( circles ) are indicated . Scale bars , 20 μm .

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CHeLa cells, transfected with GFP‐parkin (green) and siRNA targeting Drp1 and Atg5, were treated with 25 μM antimycin A for 90 min, then fixed, immunostained for PDH (red) and TOM20 (blue), and counterstained for Hoescht (gray). PDH E2/E3bp bp‐positive/TOM20‐negative MDVs colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm.


D of PDH E2 / E3 bp ‐ positive / TOM20 ‐ negative vesicles from HeLa transfected with indicated siRNA ; both total number ( bars ) number colocalizing with GFP ‐ parkin ( gray bars ) are indicated . Error bars represent mean ± s . e . m . P ‐ values are given for total vesicle number ( n = 42 - 64 in 2 - 3 experiments ) ; ns , not significant .

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D Quantification of PDH E2/E3 bp‐positive/TOM20‐negative vesicles from HeLa cells transfected with the indicated siRNA; both the total number (white bars) and the number colocalizing with GFP‐parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P‐values are given for the total vesicle number (n = 42-64 cells in 2-3 experiments); ns, not significant.


A Representative immunoblot of whole ‐ lysates from HeLa transfected with non ‐ targeting siRNA or siRNA targeting PINK1 ( siPINK1 ) , treated with 10 μM CCCP for 6 h in order to stabilize PINK1 full ‐ band .

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A Representative immunoblot of whole‐cell lysates from HeLa cells transfected with non‐targeting siRNA or siRNA targeting PINK1 (siPINK1), treated with 10 μM CCCP for 6 h in order to stabilize the PINK1 full‐length band.


B HeLa transfected with GFP − parkin ( green ) siRNA targeting PINK1 ( siPINK1 ) or non − targeting control ( ctrl siRNA ) , were treated with 25 μM antimycin A for 90 min , then fixed immunostained for PDH E2 / E3 bp ( red ) TOM20 ( blue ) . PDH E2 / E3 bp − positive / TOM20 − negative MDVs colocalizing with GFP − parkin ( arrows ) or not ( circles ) are indicated . Arrowheads indicate parkin − positive MDVs adjacent to mitochondria , possibly budding . Scale bar , 20 μm .

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B HeLa cells transfected with GFP−parkin (green) and siRNA targeting PINK1 (siPINK1) or non−targeting control (ctrl siRNA), were treated with 25 μM antimycin A for 90 min, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp−positive/TOM20−negative MDVs colocalizing with GFP−parkin (arrows) or not (circles) are indicated. Arrowheads indicate parkin−positive MDVs adjacent to mitochondria, possibly budding. Scale bar, 20 μm.


C of PDHE2 / E3 bp − positive / TOM20 − negative structures from ( B ) ; both total number ( bars ) number colocalizing with GFP − parkin ( gray bars ) are indicated . Error bars represent mean ± s . e . m . P − values are given first for GFP − / GFP − parkin − positive vesicles , then for total vesicle number ( n = 42 - 60 in three experiments ) ; ns , not significant ; * * * P < 0 . 001 ) .

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C Quantification of PDHE2/E3 bp−positive/TOM20−negative structures from (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 42-60 cells in three experiments); ns, not significant; ***P < 0.001).


A of TMRM by flow cytometry in U2OS : GFP ‐ parkin treated with 25 μM antimycin A ( anti A ) , 25 μM antimycin A 10 μM oligomycin ( anti A + oligo ) , 20 μM CCCP ( CCCP ) for indicated time , represented as a fraction of compared to DMSO ‐ treated . Error bars represent mean ± s . e . m . ( n = 3 experiments ) .

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A Quantification of TMRM fluorescence by flow cytometry in U2OS:GFP‐parkin cells treated with 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), and 20 μM CCCP (CCCP) for the indicated time, represented as a fraction of fluorescence intensity compared to DMSO‐treated cells. Error bars represent the mean ± s.e.m. (n = 3 experiments).


B U2OS : GFP ‐ parkin were treated with DMSO ( DMSO ) , 25 μM antimycin A ( anti A ) , 25 μM antimycin A 10 μM oligomycin ( anti A + oligo ) , or 20 μM CCCP ( CCCP ) for two hours , then fixed immunostained for TOM20 ( red ) . Scale bars , 50 μm .

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B U2OS:GFP‐parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for TOM20 (red). Scale bars, 50 μm.


C ofPDHE2 / E3 bp − positive / TOM20 − negative structures from ( B ) ; both total number ( bars ) number colocalizing with GFP − parkin ( gray bars ) are indicated . Error bars represent mean ± s . e . m . P − values are given first for GFP − / GFP − parkin − positive vesicles , then for total vesicle number ( n = 42 - 60 in three experiments ) ; ns , not significant ; * * * P < 0 . 001 ) .

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C Quantification ofPDHE2/E3 bp−positive/TOM20−negative structures from (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 42-60 cells in three experiments); ns, not significant; ***P < 0.001).


D U2OS : GFP − parkin were treated with DMSO ( DMSO ) , 25 μM antimycin A ( anti A ) , 25 μM antimycin A 10 μM oligomycin ( anti A + oligo ) , or 20 μM CCCP ( CCCP ) for two hours , then fixed immunostained for PDH E2 / E3 bp ( red ) TOM20 ( blue ) . A merged image of both mitochondrial markers ( mito ) is shown at far right . Arrows indicate PDH E2 / E3 bp − positive / TOM20 − negative structures colocalizing with GFP − parkin ( green ) . Scale bars , 20 μm .

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D U2OS:GFP−parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). A merged image of both mitochondrial markers (mito) is shown at the far right. Arrows indicate PDH E2/E3 bp−positive/TOM20−negative structures colocalizing with GFP−parkin (green). Scale bars, 20 μm.


A Representative immunoblot of whole ‐ lysates from U2OS : GFP GFP ‐ parkin treated with DMSO , 25 μM antimycin A ( anti A ) , 25 μM antimycin A with 10 μM oligomycin ( anti A + oligo ) , or 20 μM CCCP for indicated time period .

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A Representative immunoblot of whole‐cell lysates from U2OS:GFP and GFP‐parkin cells treated with DMSO, 25 μM antimycin A (anti A), 25 μM antimycin A with 10 μM oligomycin (anti A + oligo), or 20 μM CCCP for the indicated time period.


B of PDH E1a signal relative to that of actin in immunoblots from ( A ) . Error bars represent mean ± s . e . m . ( n = 3 experiments ) .

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B Quantification of PDH E1a signal intensity relative to that of actin in immunoblots from (A). Error bars represent the mean ± s.e.m. (n = 3 experiments).


C of mitochondrial clearance in U2OS : GFP − parkin treated as in ( A ) , fixed immunostained for TOM20 . Data are shown as of containing mitochondria , by TOM20 , visualized by microscopy . Error bars represent mean ± s . e . m . ( n = 3 experiments , with at least 85 quantified per condition , per experiment ) .

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C Quantification of mitochondrial clearance in U2OS:GFP−parkin cells treated as in (A), fixed and immunostained for TOM20. Data are shown as percentage of cells containing mitochondria, by TOM20 staining, visualized by fluorescence microscopy. Error bars represent the mean ± s.e.m. (n = 3 experiments, with at least 85 cells quantified per condition, per experiment).


D U2OS : GFP ‐ parkin were treated as in ( A ) for indicated time period , then fixed immunostained for TOM20 ( red ) . boundaries are delineated in single ‐ channel . Scale bars , 20 μm .

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D U2OS:GFP‐parkin cells were treated as in (A) for the indicated time period, then fixed and immunostained for TOM20 (red). Cell boundaries are delineated in single‐channel images. Scale bars, 20 μm.


( A ) Representative photomicrographs of MAP2 ( green ) caspase - 3 ( red ) immunocytochemistry of embryonic cortical neurons from wild type ( upper panels ) or C259A mice treated with vehicle or 10µM Aβ oligomers for 14h . Scale bar 15µm .

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(A) Representative photomicrographs of MAP2 (green) and cleaved caspase-3 (red) immunocytochemistry of embryonic cortical neurons from wild type (upper panels) or C259A mice treated with vehicle or 10µM Aβ oligomers for 14h. Scale bar 15µm.


( B ) of active ( ) caspase - 3 in cultured cortical neurons of indicated genotypes treated for 14h with vehicle or Aβ oligomers ( 2 . 5 , 5 , or 10 µM ) . Data are expressed as caspase - 3 MAP2 double positive over total number of MAP2 positive .

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(B) Quantification of active (cleaved) caspase-3 in cultured cortical neurons of the indicated genotypes treated for 14h with vehicle or Aβ oligomers (2.5, 5, or 10 µM). Data are expressed as percentage cleaved caspase-3 and MAP2 double positive cells over the total number of MAP2 positive cells.


( C ) of neurite in in cultured cortical neurons of indicated genotypes treated for 24h with vehicle or Aβ oligomers ( 2 . 5 , or 10 µM ) . Data are expressed as mean neurite in neurons , relative to wild type vehicle ( set to 100 units ) .

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(C) Quantification of neurite length in in cultured cortical neurons of the indicated genotypes treated for 24h with vehicle or Aβ oligomers (2.5, or 10 µM). Data are expressed as mean neurite length in individual neurons, relative to wild type vehicle (set to 100 units).


( A ) Immunohistochemistry for p75NTR in hippocampal CA1 of 5 month wild type , 5xFAD p75NTR knock - out ( KO ) mice . Scale bar , 20μm .

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(A) Immunohistochemistry for p75NTR in hippocampal CA1 of 5 month wild type, 5xFAD and p75NTR knock-out (KO) mice. Scale bar, 20μm.


Western blot of p75NTR expression in total lysates of hippocampus from 6 month old wild type ( WT ) 5xFAD mice assessed using antibody GT15057 against p75NTR extracellular domain lower panel shows reprobing for GAPDH to control for equal gel loading . ( C ) of p75NTR expression in mouse hippocampus relative to GAPDH .

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Western blot analysis of p75NTR expression in total lysates of hippocampus from 6 month old wild type (WT) and 5xFAD mice assessed using antibody GT15057 against p75NTR extracellular domain The lower panel shows reprobing for GAPDH to control for equal gel loading. (C) Quantification of p75NTR expression in mouse hippocampus relative to GAPDH.


( D ) Immunostaining for Aβ plaques with 6E10 antibody in coronal sections through hippocampus of 5xFAD , 5xFAD / KO , 5xFAD / ΔDD 5xFAD / C259A mice of indicated ages . Scale bar , 400μm . ( E ) of Aβ plaque burden in hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows of hippocampal occupied by Aβ plaques ( mean ± , N = 5 mice per group ) . * , P < 0 . 05 * * , P < 0 . 01 vs 5XFAD . # , P < 0 . 05 vs 5XFAD / KO ( one - way ANOVA followed by post hoc ) . ( F ) of number of Aβ plaques larger than 30um in diameter per um2 in coronal sections through hippocampus of 9 month old 5xFAD , 5xFAD / KO , 5xFAD / ΔDD 5xFAD / C259A mice . Color codes are as in panel ( E ) . Histogram shows mean ± , N = 4 mice per group ; * , P < 0 . 05 KO vs . knock - in genotypes .

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(D) Immunostaining for Aβ plaques with 6E10 antibody in coronal sections through the hippocampus of 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice of the indicated ages. Scale bar, 400μm. (E) Quantification of Aβ plaque burden in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Aβ plaques (mean ± SEM, N=5 mice per group). *, P<0.05 and **, P<0.01 vs 5XFAD. #, P<0.05 vs 5XFAD/KO (one-way ANOVA followed by post hoc test). (F) Quantification of the number of Aβ plaques larger than 30um in diameter per um2 in coronal sections through the hippocampus of 9 month old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Color codes are as in panel (E). Histogram shows mean ± SEM, N=4 mice per group; *, P<0.05 KO vs. knock-in genotypes.


ELISA determinations of Aβ1 - 42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ monomers ( G ) refers to soluble fraction after Tris - buffered saline

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ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ monomers (G) refers to the soluble fraction after Tris-buffered saline extraction


ELISA determinations of Aβ1 - 42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ oligomers ( H ) to soluble fraction after RIPA buffer extraction of Tris - buffered saline pellet ,

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ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ oligomers (H) to the soluble fraction after RIPA buffer extraction of the Tris-buffered saline pellet,


ELISA determinations of Aβ1 - 42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ fibrils ( I ) to soluble fraction after formic acid treatment of RIPA pellet .

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ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ fibrils (I) to the soluble fraction after formic acid treatment of the RIPA pellet.


( A ) Immunostaining for Glial fibrillary acidic ( GFAP ) , a marker of astrocytes , Aβ plaques in coronal sections through hippocampus of 6 month old wild type , 5xFAD , 5xFAD / KO , 5xFAD / ΔDD 5xFAD / C259A mice . Scale bar , 300μm . Right panels show high magnification of indicated areas . Scale bar , 50μm . ( B ) of GFAP in hippocampus of wild type 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows of hippocampal occupied by GFAP immunostaining ( mean ± , N = 5 mice per group ) .

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(A) Immunostaining for Glial fibrillary acidic protein (GFAP), a marker of astrocytes, and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 300μm. Right hand panels show high magnification of the indicated areas. Scale bar, 50μm. (B) Quantification of GFAP area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by GFAP immunostaining (mean ± SEM, N=5 mice per group).


( C ) Immunostaining for Ionized calcium binding adaptor molecule 1 ( Iba1 ) , a marker of microglial , Aβ plaques in coronal sections through hippocampus of 6 month old wild type , 5xFAD , 5xFAD / KO , 5xFAD / ΔDD 5xFAD / C259A . Scale bar , 300μm . Right panels show high magnification of indicated areas . Scale bar , 10μm . ( D ) of Iba1 in hippocampus of wild type 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows of hippocampal occupied by Iba1 immunostaining ( mean ± , N = 5 mice per group ) .

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(C) Immunostaining for Ionized calcium binding adaptor molecule 1 (Iba1), a marker of microglial cells, and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A. Scale bar, 300μm. Right hand panels show high magnification of the indicated areas. Scale bar, 10μm. (D) Quantification of Iba1 area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Iba1 immunostaining (mean ± SEM, N=5 mice per group).


( E ) Immunostaining of reticulon 3 ( RTN3 ) , a marker of dystrophic neurites , Aβ plaques in coronal sections through hippocampus of 6 month old 5xFAD , 5xFAD / KO , 5xFAD / ΔDD 5xFAD / C259A mice . Scale bar , 40μm . ( F ) of RTN3 - positive dystrophic neurite in hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows of Aβ plaque that overlapped with RTN3 immunostaining ( mean ± , N = 5 mice per group ) .

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(E) Immunostaining of reticulon 3 (RTN3), a marker of dystrophic neurites, and Aβ plaques in coronal sections through the hippocampus of 6 month old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 40μm. (F) Quantification of RTN3-positive dystrophic neurite area in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of Aβ plaque area that overlapped with RTN3 immunostaining (mean ± SEM, N=5 mice per group).


( G ) MitoSox , a mitochondrial superoxide indicator , DAPI in coronal sections through hippocampus of 6 month old wild type , 5xFAD , 5xFAD / KO , 5xFAD / ΔDD 5xFAD / C259A mice . Scale bar , 60μm . ( H ) of MitoSOX signal in pyramidal layer of hippocampus of wild type 5xFAD mouse strains carrying different p75NTR variants as indicated . Histogram shows MitoSox mean in arbitrary units ( mean ± , N = 5 mice per group ) .

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(G) MitoSox staining, a mitochondrial superoxide indicator, and DAPI in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 60μm. (H) Quantification of MitoSOX signal in the pyramidal cell layer of hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows MitoSox mean fluorescence intensity in arbitrary units (mean ± SEM, N=5 mice per group).


( A ) of change in field excitatory postsynaptic potential ( fEPSP ) recorded after theta - burst stimulation ( TBS ) in Schaffer collaterals of hippocampal slices from 6 month old wild type , 5xFAD 5xFAD / p75NTR mutant mice , as indicated . Results are presented as mean % change normalized to t = 0 ± . N = 6 ( wild type ) , 5 ( 5xFAD ) , 9 ( 5xFAD / KO ) , 7 ( 5xFAD / ΔDD ) 8 ( 5xFAD / C259A ) slices from 3 mice per genotype , respectively . ( B ) of fEPSP ( mean % change ± ) in indicated genotypes at 3 time points . * , P < 0 . 05 ; * * , P < 0 . 01 vs . 5xFAD ( two - way ANOVA followed by post hoc ) . N numbers as in ( A ) .

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(A) Percentage of change in field excitatory postsynaptic potential (fEPSP) recorded after theta-burst stimulation (TBS) in Schaffer collaterals of hippocampal slices from 6 month old wild type, 5xFAD and 5xFAD/p75NTR mutant mice, as indicated. Results are presented as mean % change normalized to t=0 ± SEM. N=6 (wild type), 5 (5xFAD), 9 (5xFAD/KO), 7 (5xFAD/ΔDD) and 8 (5xFAD/C259A) slices from 3 mice per genotype, respectively. (B) Quantification of fEPSP (mean % change ± SEM) in the indicated genotypes at 3 time points. *, P<0.05; **, P<0.01 vs. 5xFAD (two-way ANOVA followed by post hoc test). N numbers as in (A).


( C ) Behavior in novel object recognition ( NOR ) of 6 month old wild type , 5XFAD 5XFAD / p75NTR mutant mice , as indicated . Histograms show mean recognition index ± during training , 30min , 24h 14 days after training , corresponding to measures of short term , long term remote memory , respectively . Bar color codes are as in panel ( A ) . * , P < 0 . 05 vs . 5xFAD ( two - way ANOVA followed by post hoc ) . N = 12 ( wild type , 5xFAD / ΔDD 5xFAD / C259A ) , 10 ( 5xFAD ) 8 ( 5xFAD / KO ) mice per genotype , respectively .

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(C) Behavior in the novel object recognition (NOR) test of 6 month old wild type, 5XFAD and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean recognition index ± SEM during training, and 30min, 24h and 14 days after training, corresponding to measures of short term, long term and remote memory, respectively. Bar color codes are as in panel (A). *, P<0.05 vs. 5xFAD (two-way ANOVA followed by post hoc test). N=12 (wild type, 5xFAD/ΔDD and 5xFAD/C259A), 10 (5xFAD) and 8 (5xFAD/KO) mice per genotype, respectively.


( D ) Training latency in Barnes maze of 6 month old wild type , 5XFAD 5XFAD / p75NTR mutant mice , as indicated . Histograms show mean latency in seconds to find platform hole ± in 4 consecutive training sessions . Bar color codes are as in panel ( A ) . * , P < 0 . 05 vs . 5xFAD ; # , P < 0 . 05 vs . wild type , 5xFAD / ΔDD or 5xFAD / C259A ( two - way ANOVA followed by post hoc ) . N = 14 ( wild type 5xFAD / ΔDD ) , 10 ( 5xFAD , ( 5xFAD / KO 5xFAD / C259A ) mice per genotype , respectively .

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(D) Training latency in the Barnes maze test of 6 month old wild type, 5XFAD and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean latency in seconds to find the platform hole ± SEM in 4 consecutive training sessions. Bar color codes are as in panel (A). *, P<0.05 vs. 5xFAD; #, P<0.05 vs. wild type, 5xFAD/ΔDD or 5xFAD/C259A (two-way ANOVA followed by post hoc test). N=14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, (5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively.


N = 14 ( wild type 5xFAD / ΔDD ) , 10 ( 5xFAD , ( 5xFAD / KO 5xFAD / C259A ) mice per genotype , respectively . ( E ) of time spent ( mean ± ) in target quadrant of Barnes maze 30min , 24h 14 days after training . * , P < 0 . 05 vs . 5xFAD ; # , P < 0 . 05 vs . wild type , 5xFAD / ΔDD or 5xFAD / C259A ( two way ANOVA followed by post hoc ) .

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N=14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, (5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively. (E) Percentage of time spent (mean ± SEM) in the target quadrant of the Barnes maze test 30min, 24h and 14 days after training. *, P<0.05 vs. 5xFAD; #, P<0.05 vs. wild type, 5xFAD/ΔDD or 5xFAD/C259A (two way ANOVA followed by post hoc test).


( A ) Western blot of CTF beta ( CTFβ ) in hippocampal lysates of 9 month old 5xFAD mice carrying different p75NTR alleles detected using anti - APP - CTF antibody Arrows point to phospho - C99 C99 CTFβ species Asterisk denotes different species of native phosphorylated alpha beta CTFs of lower molecular weights Lower panel shows reprobing for GAPDH . ( B ) ( mean ± ) of phospho - C99 / C99 CTFβ species , normalized to GAPDH expressed relative to in 5xFAD mice . * , P < 0 . 05 5xFAD vs , 5xFAD / KO ; * * * , P < 0 . 001 5xFAD vs . 5xFAD / ΔDD 5xFAD / C259A ; # , P < 0 . 05 5xFAD / C259A vs . 5xFAD / KO ; § , P = 0 . 056 5xFAD / ΔDD vs . 5xFAD / KO . N = 5 mice per group .

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(A) Western blot analysis of CTF beta (CTFβ) in hippocampal lysates of 9 month old 5xFAD mice carrying different p75NTR alleles detected using anti-APP-CTF antibody Arrows point to phospho-C99 and C99 CTFβ species Asterisk denotes different species of native and phosphorylated alpha and beta CTFs of lower molecular weights Lower panel shows reprobing for GAPDH. (B) Quantification (mean ± SEM) of phospho-C99/C99 CTFβ species, normalized to GAPDH and expressed relative to levels in 5xFAD mice. *, P<0.05 5xFAD vs, 5xFAD/KO; ***, P<0.001 5xFAD vs. 5xFAD/ΔDD and 5xFAD/C259A; #, P<0.05 5xFAD/C259A vs. 5xFAD/KO; §, P=0.056 5xFAD/ΔDD vs. 5xFAD/KO. N=5 mice per group.


( C ) Western blot of soluble APP alpha ( sAPPα ) in soluble Tris - buffered saline fraction of hippocampal homogenate made from 9 moth old 5xFAD mice carrying different p75NTR alleles detected using 6E10 antibody Lower panel shows reprobing for GAPDH . ( D ) ( mean ± ) normalized to GAPDH expressed relative to in 5xFAD mice . * , P < 0 . 05 5xFAD vs . 5xFAD / KO , 5xFAD / ΔDD 5xFAD / C259A ; # , P < 0 . 05 5xFAD / C259A vs . 5xFAD / KO ; § , P < 0 . 05 5xFAD / ΔDD vs . 5xFAD / KO . N = 9 mice per group .

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(C) Western blot analysis of soluble APP alpha (sAPPα) in the soluble Tris-buffered saline fraction of hippocampal homogenate made from 9 moth old 5xFAD mice carrying different p75NTR alleles detected using 6E10 antibody Lower panel shows reprobing for GAPDH. (D) Quantification (mean ± SEM) normalized to GAPDH and expressed relative to levels in 5xFAD mice. *, P<0.05 5xFAD vs. 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A; #, P<0.05 5xFAD/C259A vs. 5xFAD/KO; §, P<0.05 5xFAD/ΔDD vs. 5xFAD/KO. N=9 mice per group.


( A ) Internalization of triple mutant hAPP in wild type , p75NTR knock - out ( KO ) , ΔDD C259A hippocampal neurons ( 15min time point ) . Counterstaining for p75NTR DAPI are also shown . Scale bar , 10μm .

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(A) Internalization of triple mutant hAPP in wild type, p75NTR knock-out (KO), ΔDD and C259A hippocampal neurons (15min time point). Counterstaining for p75NTR and DAPI are also shown. Scale bar, 10μm.