( A ) Representative colonies formed by Sirt7 + / + , Sirt7 - / - Sirt7 - TG bulge SCs that were puriﬁed from telogen - phase HFs allowed to grow for 2 . ( B - C ) Quantiﬁcations of colony number in ( A ) ; n = 3 mice per genotype .

O O O O O O O B-CONTROLLED_VAR 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 O 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) Representative colonies formed by Sirt7+/+, Sirt7-/- and Sirt7-TG bulge SCs that were puriﬁed from telogen-phase HFs and allowed to grow for 2 weeks. (B-C) Quantiﬁcations of colony number and size in (A); n = 3 mice per genotype.


Immunoblot of indicated in HFSC colonies from Sirt7 + / + , Sirt7 - / - Sirt7 - TG mice ( D )

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

Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice (D)


in HFSC colonies from Sirt7 + / + , Sirt7 - / - Sirt7 - TG mice Nfatc1 were quantified using Image J Plus 6 . 0 ( E , n = 3 mice for each group ) .

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

proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice Nfatc1 protein levels were quantified using Image J Plus 6.0 (E, n = 3 mice for each group).


( F ) Immunoblot of NFATc1 expression level in human keratinocyte HaCaT treated with SIRT7 siRNAs .

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

(F) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs.


( G ) Immunoblot of NFATc1 expression in presence of ectopic SIRT7 expression in HaCaT .

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

(G) Immunoblot analysis of the NFATc1 expression in the presence of ectopic SIRT7 expression in HaCaT cells.


( H ) NFATc1 luciferase activity was monitored in HEK293T after indicated treatments ( n = 3 ) . relative luciferase activity was determined by dual luciferase reporter .

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

(H) NFATc1 luciferase activity was monitored in HEK293T cells after the indicated treatments (n=3). The relative luciferase activity was determined by dual luciferase reporter assay.


( I - J ) analyses of Nfatc1 expression in hair follicles of Sirt7f / f , Sirt7f / f ; K15 - Cre Sirt7 - TG mice at indicted time points . black dashed indicates bulge ( Bu ) of hair follicle .

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

(I-J) IHC analyses of Nfatc1 expression in hair follicles of Sirt7f/f, Sirt7f/f ;K15-Cre and Sirt7-TG mice at the indicted time points. The black dashed line indicates the bulge (Bu) of hair follicle.


( K - L ) qPCR of Nfatc1 - targeted genes in HFSCs colonies cultured from Sirt7 + / + , Sirt7 - / - Sirt7 - TG mice , n = 3 for each genotypes .

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

(K-L) qPCR analysis of Nfatc1-targeted genes in HFSCs colonies cultured from Sirt7+/+, Sirt7-/-and Sirt7-TG mice, n=3 for each genotypes.


( A - B ) Immunoblot of interactions between ectopic HA - SIRT7 FLAG - NFATc1in HEK293T .

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

(A-B) Immunoblot analysis of the interactions between ectopic HA-SIRT7 and FLAG-NFATc1in HEK293T cells.


( C ) Co - immunoprecipitation ( CoIP ) between endogenous NFATc1 SIRT7 in HEK293T .

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

(C) Co-immunoprecipitation (CoIP) between endogenous NFATc1 and SIRT7 in HEK293T cells.


( D ) Immunoblots showing endogenous NFATc1 pull - down by GST - SIRT7 in HEK293T .

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

(D) Immunoblots showing endogenous NFATc1 pull-down by GST-SIRT7 in HEK293T cells.


( E ) Immunoblot of HA - SIRT7 WT various mutants in anti His - NFATc1 immunoprecipitates . ∆ N indicates N - terminal deletion ; ∆ C indicates C - terminal deletion ; AC indicates N - terminal C - terminal deletion .

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

(E) Immunoblot analysis of HA-SIRT7 WT and various mutants in the anti His-NFATc1 immunoprecipitates. ∆N indicates N-terminal deletion; ∆C indicates C-terminal deletion; AC indicates N-terminal and C-terminal deletion.


( F ) Immunoblot of HA - SIRT7 in truncated FLAG - NFATc1 immunoprecipitates . N indicates N - terminal domain ; C indicates C - terminal domain .

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

(F) Immunoblot analysis of HA-SIRT7 levels in truncated FLAG-NFATc1 immunoprecipitates. N indicates the N-terminal domain; C indicates C-terminal domain.


( G - H ) Immunoblot of interactions between endogenous NFATc1 SIRT7 in keratinocyte HaCaT .

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

(G-H) Immunoblot analysis of the interactions between endogenous NFATc1 and SIRT7 in keratinocyte HaCaT cells.


( I - J ) Immunoblot of NFATc1 acetylation in response to indicated treatments .

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

(I-J) Immunoblot analysis of NFATc1 acetylation levels in response to the indicated treatments.


Immunoblot of NFATc1 - WT / K612R acetylation on SIRT7 deacetylation assays in vitro ( K )

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

Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vitro (K)


Immunoblot of NFATc1 - WT / K612R acetylation on SIRT7 deacetylation assays in vivo ( L ) .

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

Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vivo (L).


Immunoblots showing NFATc1 pan phosphorylation ( probing to anti S / T antibody ) acetylation under indicated treatments .

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

Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments.


( O ) Immunoblot of NFATc1 phosphorylation among K612 mutants , noting that K612R represents deacetylation while K612Q hyperacetylation .

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

(O) Immunoblot analysis of NFATc1 phosphorylation among K612 mutants, noting that K612R represents deacetylation while K612Q hyperacetylation.


( A ) Representative immunoblots showing NFATc1 degradation in HEK293T stably overexpressing NFATc1 in presence of cycloheximide ( CHX ) , with or without SIRT7 overexpression . ( B ) of NFATc1 in ( A ) from 3 independent experiments .

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

(A) Representative immunoblots showing NFATc1 degradation in HEK293T cells stably overexpressing NFATc1 in the presence of cycloheximide (CHX), with or without SIRT7 overexpression. (B) Quantification of NFATc1 levels in (A) from 3 independent experiments.


( C ) Immunoblots of Nfatc1 in Sirt7 + / + Sirt7 - / - keratinocytes . ( D ) of Nfatc1 in ( C ) from 3 independent experiments .

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

(C) Immunoblots of Nfatc1 protein levels in Sirt7+/+ and Sirt7-/- keratinocytes. (D) Quantification of Nfatc1 levels in (C) from 3 independent experiments.


( E - F ) Immunoblots ( E ) related quantitative data ( F , n = 3 ) showing NFATc1 in HEK293T overexpressing wildtype HA - SIRT7 or a SIRT7 enzyme - dead mutant ( H187Y ) .

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

(E-F) Immunoblots (E) and related quantitative data (F, n=3) showing NFATc1 protein levels in HEK293T cells overexpressing wildtype HA-SIRT7 or a SIRT7 enzyme-dead mutant (H187Y).


( G - H ) Immunoblots ( G ) quantitative data ( H , n = 3 ) showing NFATc1 in HEK293T transfected with NFATc1 - CA , with or without ectopic SIRT7 , treated with MG132 .

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

(G-H) Immunoblots (G) and quantitative data (H, n=3) showing NFATc1 levels in HEK293T cells transfected with NFATc1-CA, with or without ectopic SIRT7, treated with MG132.


( I - J ) Immunoblots ( I ) related quantitative data ( J , n = 3 ) showing NFATc1 - WT NFATc1 - K612R turnover in HEK293T .

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

(I-J) Immunoblots (I) and related quantitative data (J, n=3) showing NFATc1-WT and NFATc1-K612R protein turnover in HEK293T cells.


Immunoblots showing interaction ( by co - immunoprecipitation ) between ectopic NFATc1 / PA28γ in HEK293T ( K ) ( L ) after manipulating SIRT7 .

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 B-CONTROLLED_VAR O

Immunoblots showing the interaction (by co-immunoprecipitation) between ectopic NFATc1/PA28γ in HEK293T cells (K) (L) after manipulating SIRT7 levels.


Immunoblots showing interaction ( by co - immunoprecipitation ) between endogenous NFATc1 / PA28γ in HaCaT ( L ) after manipulating SIRT7 .

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 B-CONTROLLED_VAR O

Immunoblots showing the interaction (by co-immunoprecipitation) between endogenous NFATc1/PA28γ in HaCaT cells (L) after manipulating SIRT7 levels.


( M - N ) Immunoblot ( M ) quantitative data ( N , n = 3 ) of NFATc1 upon overexpression of HA - SIRT7 with or without PA28γ oligo siRNAs treatment .

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

(M-N) Immunoblot (M) and quantitative data (N, n=3) analysis of NFATc1 levels upon overexpression of HA-SIRT7 with or without PA28γ oligo siRNAs treatment.


( O ) Co - immunoprecipitation between NFATc1 PA28γ in HEK293T cotransfected with WT HA - SIRT7 or an enzyme - dead mutant ( H187Y ) .

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

(O) Co-immunoprecipitation between NFATc1 and PA28γ in HEK293T cells cotransfected with WT HA-SIRT7 or an enzyme-dead mutant (H187Y).


( A ) of Nfatc1 expression in HFs on P84 in Sirt7 + / + Sirt7 - / - mice with or without Cyclosporin A ( CsA ) treatment . black dashed indicates bulge ( Bu ) of hair follicle . ( B ) Quantitative of Nfatc1 in ( A , n = 10 hair follicles ) using ImageJ plus 6 . 0 .

O O O O B-MEASURED_VAR 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 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

(A) IHC staining of Nfatc1 expression in HFs on P84 in Sirt7+/+ and Sirt7-/- mice with or without Cyclosporin A (CsA) treatment. The black dashed line indicates bulge (Bu) of hair follicle. (B) Quantitative analysis of Nfatc1 levels in (A, n=10 hair follicles) using ImageJ plus 6.0.


( C ) Immunofluorescent of Ki67 Pcad at P84 in Sirt7 + / + Sirt7 - / - HFs with or without CsA treatment .

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

(C) Immunofluorescent staining of Ki67 and Pcad at P84 in Sirt7+/+ and Sirt7-/- HFs with or without CsA treatment.


( D ) of Ki67 - positive per HG in ( C , n = 10 hair follicles ) .

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

(D) The percentage of Ki67-positive cells per HG in (C, n=10 hair follicles).


( E ) H & E of skin in indicated treatment groups at P100 .

(E) H&E staining of the skin in the indicated treatment groups at P100.


( F ) Representative showing haircoat recovery after indicated treatments . dashed circle indicates shaved ; dotted indicates hair growth boundary .

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

(F) Representative images showing haircoat recovery after the indicated treatments. The dashed circle indicates the shaved area; the dotted line indicates the hair growth boundary.


( A ) Immunoblot of Sirt7 expression in young ( 3M ) aged ( 18M ) HFSCs . # 1 , # 2 , # 3 represented mice . Below , of Sirt7 ; n = 3 mice for each group ; Y - axis represents relative band ( measured with Image J ® ) normalized to signal in young mice .

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 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

(A) Immunoblot analysis of Sirt7 expression in young (3M) and aged (18M) HFSCs. #1, #2, #3 represented individual mice. Below, quantification of Sirt7 protein levels; n=3 mice for each group; the Y-axis represents the relative band intensity (measured with Image J®) normalized to the signal in young mice.


( B - D ) Immunofluorescence of Nfatc1 Sirt7 in HFs of young ( 3 months ) old ( 18 months ) mice ( B ) , relative of Sirt7 Nfatc1 - positive in bulge ( C - D ) . / black dashed indicates bulge ( Bu ) of hair follicle .

O O O O O O O B-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-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

(B-D) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of young (3 months) and old (18 months) mice (B), and the relative levels of Sirt7 and Nfatc1-positive cells in the bulge (C-D). The white/black dashed line indicates bulge (Bu) of hair follicle.


( E ) were captured of hair coats of Sirt7 + / + Sirt7 - / - mice at 15 months . yellow dashed lines indicate hair thinning or hair loss .

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 O O O O

(E) Images were captured of the hair coats of Sirt7+/+ and Sirt7-/- mice at 15 months. The yellow dashed lines indicate hair thinning or hair loss.


( F ) Immunofluorescence of Nfatc1 Sirt7 in HFs of 15 - month - old Sirt7 + / + Sirt7 - / - mice . / black dashed indicates bulge ( Bu ) of hair follicle .

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

(F) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of 15-month-old Sirt7+/+ and Sirt7-/- mice. The white/black dashed line indicates bulge (Bu) of hair follicle.


( G ) of Nfatc1 - positive per bulge in ( F , n = 10 hair follicles ) .

(G) Quantification of Nfatc1-positive cells per bulge in (F, n=10 hair follicles).


( H ) captured of skin in plucked regions ( blue dotted lines ) of WT ( 3 months ) , aged WT ( 20 months ) aged Sirt7 - TG mice ( 20 months ) , 14 days after depilation . Pigmentation of skin indicates hair regrowth . n = 3 mice per genotype .

O O O O 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

(H) Images captured of the skin in the plucked regions (blue dotted lines) of WT (3 months), aged WT (20 months) and aged Sirt7-TG mice (20 months), 14 days after depilation. Pigmentation of the skin indicates hair regrowth. n = 3 mice per genotype.


( I ) Hair re - growth scores were analyzed for young WT , old WT old Sirt7 - TG mice in ( H ) , n = 3 mice for each group .

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

(I) Hair re-growth scores were analyzed for young WT, old WT and old Sirt7-TG mice in (H), n=3 mice for each group.


( J ) hair coat recovery score ( described in methods ) after back hair of old WT old Sirt7 - TG mice was shaved . n = 3 mice per genotype .

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

(J) The hair coat recovery score (described in methods) after the back hair of old WT and old Sirt7-TG mice was shaved. n = 3 mice per genotype.


( K ) captured of hair coats of WT Sirt7 - TG mice at 28 months - of - . yellow dashed lines indicate hair thinning or hair loss regions .

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

(K) Images captured of the hair coats of WT and Sirt7-TG mice at 28 months-of-age. The yellow dashed lines indicate the hair thinning or hair loss regions.


( A - J ) Confocal of different z sections ( 0 . 2 µm of thickness ) of a neuron transfected with GFP - LC3 ( A - D ) sections are flattened ( E ) . Higher magnification of outlined in are shown in inserts ( A - D ) . Different sections of of transfected neuron with GFP - LC3 ( green ) merged with DAPI ( blue ) . Scale bar , 20 µm . ( K - O ) Three - dimensional reconstruction from serial sections using IMARIS software , process ( K ) higher magnification of zoomed outlined in ( K - I ) . Reconstructed 3D image of same neuron using IMARIS , which evaluates AV localization in reconstructed 3D image ( M - O ) . Enlarged areas of neuron revealed presence of AV in soma neurites ( N - O ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Confocal images of different z sections (0.2 µm of thickness) of a neuron transfected with GFP-LC3 (A-D) and all sections are flattened (E). Higher magnification images of the area outlined in white are shown in the inserts (A-D). Different sections of images of transfected neuron with GFP-LC3 (green) merged with DAPI (blue). Scale bar, 20 µm. (K-O) Three-dimensional reconstruction from serial sections using IMARIS software, cell body and process (K) and higher magnification of the zoomed area outlined in white (K-I). Reconstructed 3D image of the same neuron using IMARIS, which evaluates AV localization in the reconstructed 3D image (M-O). Enlarged areas of neuron revealed the presence of AV in soma and neurites (N-O).


( B ) Total AV counting results show that a significant drop in number of AV occurs in neurons exposed to SIV - infected microglia supernatant for 3 or 24 hr . Total number of AV are significantly increased after pretreatment with rapamycin followed by SIV - infected microglia supernatant ( Sup ) exposure for 24 hr . * * * P < 0 . 001 , * P < 0 . 05 for n = 6 experiments . values are mean ± . AV counting using 3D model reconstruction for neurons exposed to different treatments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV-infected microglia supernatant for 3 or 24 hr. Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV-infected microglia supernatant (Sup) exposure for 24 hr. *** P<0.001, * P<0.05 for n = 6 experiments. All values are mean±SEM. AV counting using 3D model reconstruction for neurons exposed to different treatments.


( C ) Flattened of multi - stack confocal optical slices of neurons transfected with GFP - LC3 ( green ) to label AV , DAPI ( blue ) to label nucleus . five panels display a typical sample image of a neuron under control conditions as well as 3 hr 24 hr exposure to SIV - infected microglia supernatant or pretreated with rapamycin prior exposure to SIV - infected microglia supernatant . right panels show a snap shot of a 3D outline of neuron with AV marked as green spheres . Scale bar , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV-infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV-infected microglia supernatant. The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres. Scale bar, 20 µm.


( A ) number of AV in neurites decreased significantly in neurons exposed to SIV - infected microglia supernatant for 3 or 24 hr , however ; pretreatment with rapamycin blocked this effect . * * * P < 0 . 001 , for n = 6 experiments . Scale bar , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 number of AV in neurites decreased significantly in neurons exposed to the SIV-infected microglia supernatant for 3 or 24 hr, however; pretreatment with rapamycin blocked this effect. *** P<0.001, for n = 6 experiments. Scale bar, 20 µm.


( B ) AV number is unchanged in neuronal soma after exposure to SIV - infected microglia supernatant for 3 or 24 hr .

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) The AV number is unchanged in neuronal soma after exposure to the SIV-infected microglia supernatant for 3 or 24 hr.


( C D ) Both total of neuronal processes ( C ) number of brunch points ( D ) are decreased after exposure to after SIV - infected microglia supernatant treatment . * * * P < 0 . 001 , for n = 6 experiments . values are mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) Both the total length of neuronal processes (C) and number of brunch points (D) are decreased after exposure to after SIV-infected microglia supernatant treatment. *** P<0.001, for n = 6 experiments. All values are mean±SEM.


( A ) LC3 - II level is reduced after 3 or 24 hr exposure to SIV - infected microglia supernatant this decrease effect is blocked when is pretreated with rapamycin ( 2 µM ) . level of elongation Atg12 - Atg5 is also reduced , again effect is blocked in presence of rapamycin . level of p62 is increased for similar conditions with SIV - infected microglia supernatant , increase is blocked in presence of rapamycin . GAPDH was used in these experiments as loading control . One representative experiment of n = 4 is shown .

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 B-MEASURED_VAR 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 O O O O O O O O O O O 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 LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown.


( B , C D ) Ratios between LC3 - II , Atg12 - Atg5 , p62 , respectively , normalized to GAPDH . Data are reported as mean ± ( n = 4 ) . * * * P < 0 . 001 .

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

(B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.


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 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


( A ) Total AV counting results show that a significant drop occurs in neurons exposed to TNF - α or NMDA for 3 or 24 hr . * * * P < 0 . 001 , * * P < 0 . 01 for n = 6 experiments . values are mean ± . ( B C ) Distribution of AV number in soma neurites respectively .

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

(A) Total AV counting results show that a significant drop occurs in neurons exposed to TNF-α or NMDA for 3 or 24 hr. *** P<0.001, ** P<0.01 for n = 6 experiments. All values are mean±SEM. (B and C) Distribution of AV number in soma and neurites respectively.


( B ) number of AV in neurites decreased significantly in neurons exposed to both TNF - α or NMDA for 3 or 24 hr . * * * P < 0 . 001 , for n = 6 experiments .

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

(B) The number of AV in neurites decreased significantly in neurons exposed to both TNF-α or NMDA for 3 or 24 hr. *** P<0.001, for n = 6 experiments.


( C ) AV number in soma is increased in neurons after exposure to TNF - α for 3 or 24 hr , however it remained unaffected in neurons after exposure to NMDA . * * P < 0 . 01 for n = 6 experiments .

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

(C) The AV number in soma is increased in neurons after exposure to TNF-α for 3 or 24 hr, however it remained unaffected in neurons after exposure to NMDA. ** P<0.01 for n = 6 experiments.


( D ) Flattened of multi - stack confocal optical slices of neurons transfected with GFP - LC3 ( green ) to label AV , DAPI ( blue ) to label nucleus . transfected with GFP - LC3 ( green ) to label AV , DAPI ( blue ) to label nucleus . four panels display a typical sample image of a neuron under 3 hr 24 hr exposure to TNF - α or NMDA at 25 ng / ml 35 µM respectively . Scale bar , 20 µm .

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

(D) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The four panels display a typical sample image of a neuron under 3 hr and 24 hr exposure to TNF-α or NMDA at 25 ng/ml and 35 µM respectively. Scale bar, 20 µm.


( A ) . Neuronal survival tested by MTT . There are significant decreases of neuronal survival after exposure to SIV - infected microglia supernatant for 3 or 24 hr , which is significantly abrogated in presence of rapamycin . Rapamycin alone has no effect . MK - 801 ( 100 µM ) also prevented neurotoxicity induced by SIV - infected microglia supernatant . * * * P < 0 . 001 , for n = 6 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

(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments.


( B ) Neuronal survival was examined with MTT in cultures exposed to SIV - infected microglia supernatant for 24 hr in presence or absence of different drugs such as 3 - MA ( 1 mM ) or Baf ( 100 nM ) . There is no significant difference between exposed to SIV - infected microglia supernatant a pretreatment with 3 - MA followed by exposure to SIV - infected microglia supernatant . However , there is a significant decrease of neuronal survival exposed to SIV - infected microglia supernatant when are pretreated with Baf . SIV - infected microglia supernatant from no infected monkey ( no Sup ) doesn ' t affect neuronal survival there is no significant difference with control samples . values are mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.


( A ) Relative values ( RV ) of p62 mRNA level in frontal lobe of SIVE versus SIV uninfected ( UN ) monkeys , n = 7 for SIVE , n = 12 for SIV n = 9 for UN , P < 0 . 05 .

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

(A) Relative values (RV) of p62 mRNA level in frontal lobe of SIVE versus SIV and uninfected (UN) monkeys, n = 7 for SIVE, n = 12 for SIV and n = 9 for UN, P<0.05.


B ) RV of p62 mRNA level in frontal cortex of HAD versus non neurological diseased subjects ( NNDS ) , n = 8 for HAD n = 10 for NNDS , P < 0 . 0001 .

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) RV of p62 mRNA level in frontal cortex of HAD versus non neurological diseased subjects (NNDS), n = 8 for HAD and n = 10 for NNDS, P<0.0001.


( C ) p62 ubiquitin immunoreactivity on serial sections in SIVE monkeys , as well as those with HAD in brain sections from hippocampus . p62 has a diffuse immunoreactivity in UN monkeys in human NNDS a dot profile structures in SIVE HAD brain tissues . Higher magnification views are shown in green insets . Black arrows indicate p62 - or ubiquitin - immunoreactive structures within neuronal or proximal processes , red arrowheads indicate a dot - like immunoreactivity in neuropil . Scale bar , 20 µm .

O O O B-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-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 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

(C) p62 and ubiquitin immunoreactivity on serial sections in SIVE monkeys, as well as those with HAD in brain sections from hippocampus. p62 has a diffuse immunoreactivity in UN monkeys and in human NNDS and a dot profile structures in SIVE and HAD brain tissues. Higher magnification views are shown in green insets. Black arrows indicate p62- or ubiquitin-immunoreactive structures within the neuronal cell body or proximal processes, and red arrowheads indicate a dot-like immunoreactivity in the neuropil. Scale bar, 20 µm.


IFNγ enhances macrophage host after type I ( RH ) type II ( Pru ) Toxoplasma gondii ( Tg ) infection . Graphs show as measured by LDH release ( left ) viability measured by XTT ( right ) in untreated or IFNγ - primed THP - 1 infected with type I or type II Tg for 24 h .

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-MEASURED_VAR 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

IFNγ enhances macrophage host cell death after type I (RH) and type II (Pru) Toxoplasma gondii (Tg) infection. Graphs show cell death as measured by LDH release assay (left) and viability measured by XTT assay (right) in untreated or IFNγ-primed THP-1 cells infected with type I or type II Tg for 24 h.


LDH release assays from THP - 1 left untreated or primed with IFNγ , transfected with siRNA against indicated GBPs or non - targeting control ( CTRL ) infected with indicated strains of Tg for 24 h .

B-MEASURED_VAR 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

LDH release assays from THP-1 cells left untreated or primed with IFNγ, transfected with siRNA against indicated GBPs or non-targeting control (CTRL) and infected with indicated strains of Tg for 24 h.


LDH release from monocyte - derived macrophages ( MDM ) left untreated or treated with IFNγ , transfected with siRNA against GBP1 or non - targeting control ( CTRL ) infected with indicated of Tg for 24 hours . Mean ± of n = 4 donors shown .

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

LDH release assay from primary monocyte-derived macrophages (MDM) left untreated or treated with IFNγ, transfected with siRNA against GBP1 or non-targeting control (CTRL) and infected with indicated strain of Tg for 24 hours. Mean ± SEM of n = 4 donors shown.


LDH release from indicated wildtype , GBP1 knockout ( ∆ GBP1 ) or GBP1 reconstituted ( ∆ GBP1 + Tet - GBP1 ) infected with type I or type II Tg for 24 hours . were untreated or treated with IFNγ or additionally treated with doxycycline ( Dox ) as indicated .

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

LDH release assay from indicated wildtype, GBP1 knockout (∆GBP1) or GBP1 reconstituted (∆GBP1+Tet-GBP1) cells infected with type I or type II Tg for 24 hours. Cells were untreated or treated with IFNγ or additionally treated with doxycycline (Dox) as indicated.


Realtime propidium iodide ( PI ) uptake from indicated THP - 1 infected with type I or type II Tg , untreated or treated with IFNγ / or Dox as labelled . AU , arbitrary units .

O 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

Realtime propidium iodide (PI) uptake assay from indicated THP-1 cells infected with type I or type II Tg, and untreated or treated with IFNγ and/or Dox as labelled. AU, arbitrary units.


LDH release from wildtype THP - 1 or ∆ GBP1 stably reconstituted with Dox - inducible expression plasmids of indicated mutants of GBP1 . were pre - treated with IFNγ Dox infected with either type I or type II Tg for 24 h .

B-MEASURED_VAR 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

LDH release assay from wildtype THP-1 or ∆GBP1 cells stably reconstituted with Dox-inducible expression plasmids of the indicated mutants of GBP1. Cells were pre-treated with IFNγ and Dox and infected with either type I or type II Tg for 24 h.


Propidium iodide ( PI ) uptake in indicated wildtype THP - 1 or ∆ CASP - 1 , ∆ CASP - 4 , ∆ ASC , or THP - 1 stably expressing GSDMD - targeting miRNA ( GSDMDmiR ) infected with indicated strains of Toxoplasma gondii ( Tg ) for 24 h . were untreated or treated with IFNγ as indicated . Mean under curve ( AUC ) ± from n = 3 experiments are shown . ns , not significant , from two - way ANOVA following adjustment for multiple comparisons .

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

Propidium iodide (PI) uptake in indicated wildtype THP-1 or ∆CASP-1, ∆CASP-4, ∆ASC cells, or THP-1 stably expressing GSDMD-targeting miRNA (GSDMDmiR) infected with the indicated strains of Toxoplasma gondii (Tg) for 24 h. Cells were untreated or treated with IFNγ as indicated. Mean area under the curve (AUC) ± SEM from n = 3 experiments are shown. ns, not significant, from two-way ANOVA following adjustment for multiple comparisons.


Real - time apoptosis ( Annexin V ( AnnV ) - Glo luminescence ) from IFNγ - primed human MDMs transfected with non - targeting control ( siCTRL ; left ) or GBP1 siRNA ( siGBP1 ; right ) left uninfected or infected with type I or type II Tg . Depicted is mean ± of n = 4 donors . AU , arbitrary units .

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 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

Real-time apoptosis assay (Annexin V (AnnV)-Glo luminescence) from IFNγ-primed primary human MDMs transfected with non-targeting control (siCTRL; left) or GBP1 siRNA (siGBP1; right) and left uninfected or infected with type I or type II Tg. Depicted is the mean ± SEM of n = 4 donors. AU, arbitrary units.


Immunoblots from MDMs infected with indicated Tg strains for 12 h . were untreated or pre - treated with IFNγ before infection . Treatment with TNFα cycloheximide ( CHX ) served as positive control for apoptosis . are representative of n = 4 biologically independent experiments .

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Immunoblots from primary MDMs infected with the indicated Tg strains for 12 h. Cells were untreated or pre-treated with IFNγ before infection. Treatment with TNFα and cycloheximide (CHX) served as positive control for apoptosis. Images are representative of n = 4 biologically independent experiments.


Live time - lapse imaging to visualize apoptotic measure caspase - 3 / 7 activities using a caspase substrate . THP - 1 WT were primed with IFNγ or left untreated infected with either type I or type II Tg . Plot in ( D ) shows cumulative caspase - 3 / 7 activity from an 18 - hour time - course ( mean ± from n = 3 independent experiments ) . Representative phase - contrast overlaid with signal for caspase - 3 / 7 activity ( green ) are shown in ( E ) from IFNγ - primed THP - 1 infected with type I Tg captured at indicated times post - infection . Scale bar 20 μm .

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

Live time-lapse imaging to visualize apoptotic cell death and measure caspase-3/7 activities using a fluorescent caspase substrate. THP-1 WT were primed with IFNγ or left untreated and infected with either type I or type II Tg. Plot in (D) shows cumulative caspase-3/7 activity from an 18-hour time-course (mean ± SEM from n = 3 independent experiments). Representative phase-contrast images overlaid with fluorescence signal for caspase-3/7 activity (green) images are shown in (E) from IFNγ-primed THP-1 cells infected with type I Tg captured at indicated times post-infection. Scale bar 20 μm.


AnnV - Glo from IFNγ - primed THP - 1 transfected with siRNA against indicated human caspases infected with type I or type II Tg . under curve ( AUC ) from a real - time AnnV - Glo luminescence assays similar to those in ( B ) are plotted as mean ± from n = 3 independent experiments .

B-MEASURED_VAR 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-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

AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human caspases and infected with type I or type II Tg. Area under the curve (AUC) from a real-time AnnV-Glo luminescence assays similar to those in (B) are plotted as mean ± SEM from n = 3 independent experiments.


AnnV - Glo from IFNγ - primed human MDMs transfected with siRNA against CASP8 , CASP10 non - targeting control ( CTRL ) infected with type I or type II Tg . under curve ( AUC ) from real - time assays similar to those in ( B ) are plotted from n = 4 independent experiments . Matched shapes color of symbols indicate donors .

B-MEASURED_VAR 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

AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against CASP8, CASP10 and non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays similar to those in (B) are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors.


AnnV - Glo from IFNγ - primed THP - 1 transfected with siRNA against indicated human NLR / ALR sensors infected with type I or type II Toxoplasma gondii ( Tg ) . under curve ( AUC ) from real - time assays are plotted as mean ± from n = 3 independent experiments .

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

AnnV-Glo assay from IFNγ-primed THP-1 transfected with siRNA against indicated human NLR/ALR sensors and infected with type I or type II Toxoplasma gondii (Tg). Area under the curve (AUC) from real-time assays are plotted as mean ± SEM from n = 3 independent experiments.


AnnV - Glo from IFNγ - primed human MDMs transfected with siRNA against AIM2 or non - targeting control ( CTRL ) infected with type I or type II Tg . under curve ( AUC ) from real - time assays are plotted from n = 4 independent experiments . Matched shapes color of symbols indicate donors .

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

AnnV-Glo assay from IFNγ-primed primary human MDMs transfected with siRNA against AIM2 or non-targeting control (CTRL) and infected with type I or type II Tg. Area under the curve (AUC) from real-time assays are plotted from n = 4 independent experiments. Matched shapes and color of symbols indicate donors.


Representative ( top ) from IFNγ - primed THP - 1 WT infected with type I Tg for 8 hours stained for ASC active caspase - 8 . Blue , Nuclei ; Red , ASC ; Green , active caspase - 8 ( IETD - FITC ) Grey , Tg . Scale bar , 5 μm or 2 μm in magnified . of ASC specks ( bottom ) in Tg - infected THP - 1 WT or ∆ CASP1 treated with IFNγ or left untreated at 8 hours post infection , plotted as mean ± from n = 3 independent experiment .

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

Representative images (top) from IFNγ-primed THP-1 WT infected with type I Tg for 8 hours and stained for ASC and active caspase-8. Blue, Nuclei; Red, ASC; Green, active caspase-8 (IETD-FITC) and Grey, Tg. Scale bar, 5 μm or 2 μm in the magnified images. Quantification of ASC specks (bottom) in Tg-infected THP-1 WT or ∆CASP1 treated with IFNγ or left untreated at 8 hours post infection, plotted as mean ± SEM from n = 3 independent experiment.


Immunoblots from MDMs infected with Tg strains for indicated times . were untreated or pre - treated with IFNγ before infection . are representative of n = 4 biologically independent experiments .

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

Immunoblots from primary MDMs infected with Tg strains for indicated times. Cells were untreated or pre-treated with IFNγ before infection. Images are representative of n = 4 biologically independent experiments.


Representative ( A ) ( B ) from IFNγ - primed THP - 1 ∆ GBP1 stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or monocyte derived macrophages ( MDM ) stained for GBP1 . were infected with type I or type II Toxoplasma gondii ( Tg ) for 2 h . Blue , Nuclei ; Grey , Tg ; Red , mCH - GBP1 or Magenta , GBP1 . Scale bar , 5 μm . Graph in ( B ) shows mean of Tg vacuoles targeted by GBP1 from n = 4 experiments .

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

Representative images (A) and quantification (B) from IFNγ-primed THP-1 ∆GBP1 stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or monocyte derived macrophages (MDM) stained for GBP1. Cells were infected with type I or type II Toxoplasma gondii (Tg) for 2 h. Blue, Nuclei; Grey, Tg; Red, mCH-GBP1 or Magenta, GBP1. Scale bar, 5 μm. Graph in (B) shows mean percentage of Tg vacuoles targeted by GBP1 from n = 4 experiments.


LDH release from indicated THP - 1 untreated or treated with IFNγ infected with Salmonella Typhimurium ( STm ) WT or a SPI - 1 mutant ΔinvA for 4 h at indicated multiplicities of infection ( MOI ) . Mean ± of n = 3 experiments are shown .

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

LDH release assay from indicated THP-1 cells untreated or treated with IFNγ and infected with Salmonella Typhimurium (STm) WT or a SPI-1 mutant ΔinvA for 4 h at indicated multiplicities of infection (MOI). Mean ± SEM of n = 3 experiments are shown.


Propidium iodide ( PI ) uptake from IFNγ - primed MDMs transfected with non - targeting control siRNA ( CTRL ) or siRNA against GBP1 ( left ) or indicated lines of THP - 1 ( right ) infected with STm - GFP for 4 h . under curve ( AUC ) from real - time plotted as mean ± from n = 3 independent experiments .

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

Propidium iodide (PI) uptake assay from IFNγ-primed primary MDMs transfected with non-targeting control siRNA (CTRL) or siRNA against GBP1 (left) or indicated lines of THP-1 (right) infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.


PI uptake from IFNγ - primed MDMs transfected with indicated siRNA or non - targeting control ( CTRL ) infected with STm - GFP for 4 h . under curve ( AUC ) from real - time plotted as mean ± from n = 4 independent experiments .

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

PI uptake assay from IFNγ-primed primary MDMs transfected with indicated siRNA or non-targeting control (CTRL) and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 4 independent experiments.


PI uptake from IFNγ - primed or untreated THP - 1 ΔCASP - 1 transfected with indicated siRNA infected with STm - GFP for 4 h . under curve ( AUC ) from real - time plotted as mean ± from n = 3 independent experiments . Schematic on right shows overview of pathway leading to caspase - 1 caspase - 4 activation .

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

PI uptake assay from IFNγ-primed or untreated THP-1 ΔCASP-1 cells transfected with indicated siRNA and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows overview of the pathway leading to caspase-1 and caspase-4 activation.


LDH release from indicated THP - 1 treated with IFNγ Dox infected with STm - GFP for 4 h . Shown is mean ± of n = 3 experiments .

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

LDH release assay from indicated THP-1 cells treated with IFNγ and Dox and infected with STm-GFP for 4 h. Shown is the mean ± SEM of n = 3 experiments.


Representative from IFNγ - primed THP - 1 ∆ GBP1 stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or MDMs stained for GBP1 . were infected with STm - GFP for 2 h . Blue , Nuclei ; Grey , STm ; Red , mCH - GBP1 or Magenta GBP1 . Scale bar , 5 μm .

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

Representative images from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm.


( G ) from IFNγ - primed THP - 1 ∆ GBP1 stably reconstituted with Tet - mCH - GBP1 , mCH - GBP1K51A , mCH - GBP1C589A or mCH - GBP1 ∆ 589 - 592 variants or MDMs stained for GBP1 . were infected with STm - GFP for 2 h . Blue , Nuclei ; Grey , STm ; Red , mCH - GBP1 or Magenta GBP1 . Scale bar , 5 μm . Graph in ( G ) shows of STm vacuoles targeted by GBP1 .

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

quantification (G) from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm. Graph in (G) shows percentage of STm vacuoles targeted by GBP1.


of GBP1 recruitment to STm - GFP containing vacuoles ( SCV ) in MDMs pre - treated with IFNγ or left untreated ( UT ) . Mean ± 95 % CI of GBP1 measured on indicated number ( n ) of SCVs for each condition from three independent experiments are shown .

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

Quantification of GBP1 recruitment to STm-GFP containing vacuoles (SCV) in MDMs pre-treated with IFNγ or left untreated (UT). Mean ± 95% CI of GBP1 fluorescence intensity measured on the indicated number (n) of SCVs for each condition from three independent experiments are shown.


Representative from THP - 1 ∆ GBP1 + Tet - mCH - GBP1 stably expressing YFP - CASP4C258S . were infected with STm for 2 h . Blue , Nuclei & STm ( Hoechst dye ) ; Red , mCH - GBP1 ; Green , YFP - CASP4C258S . Scale bar , 5 μm .

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

Representative images from THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S. Cells were infected with STm for 2 h. Blue, Nuclei & STm (Hoechst dye); Red, mCH-GBP1; Green, YFP-CASP4C258S. Scale bar, 5 μm.


of THP - 1 ∆ GBP1 + Tet - mCH - GBP1 stably expressing YFP - CASP4C258S infected with STm for 2 h . Graph shows of STm vacuoles targeted by GBP1 , CASP4 or both from n = 3 experiments . n . d . not detected .

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

Quantification of THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S and infected with STm for 2 h. Graph shows percentage of STm vacuoles targeted by GBP1, CASP4 or both from n = 3 experiments. n.d. not detected.


measured using Annexin V ( AnnV ) - Glo of THP - 1 ∆ CASP1 transfected with non - targeting control ( CTRL ) or GBP1 siRNA treated as indicated . were primed with IFNγ , transfected with soluble Tg antigen ( STAg ) or both , or left untreated ( UT ) . under curve ( AUC ) from real - time plotted as mean ± from n = 3 independent experiments .

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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O

Cell death measured using Annexin V (AnnV)-Glo assay of THP-1 ∆CASP1 cells transfected with non-targeting control (CTRL) or GBP1 siRNA and treated as indicated. Cells were primed with IFNγ, transfected with soluble Tg antigen (STAg) or both, or left untreated (UT). Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.


AnnV - Glo from naïve or IFNγ - primed THP - 1 ∆ CASP - 1 transfected with soluble Tg antigen ( STAg ) that was untreated ( UT ) or treated as indicated . under curve ( AUC ) from real - time plotted as mean ± from n = 3 independent experiments .

B-MEASURED_VAR 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

AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with soluble Tg antigen (STAg) that was untreated (UT) or treated as indicated. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.


Immunoblots from IFNγ - primed THP - 1 ∆ CASP - 1 or THP - 1 ∆ ASC transfected with non - targeting control ( CTRL ) , GBP1 , AIM2 , CASP - 1 or CASP - 8 siRNA then untreated ( - ) or transfected with STAg for 12 h . are representative of n = 2 independent experiments .

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

Immunoblots from IFNγ-primed THP-1∆CASP-1 or THP-1 ∆ASC cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (-) or transfected with STAg for 12 h. Images are representative of n = 2 independent experiments.


AnnV - Glo from naïve or IFNγ - primed THP - 1 ∆ CASP - 1 transfected with non - targeting control ( CTRL ) , GBP1 , AIM2 , CASP - 1 or CASP - 8 siRNA then untreated ( UT ) or transfected with STAg as indicated for 18 h . under curve ( AUC ) from real - time plotted as mean ± from n = 3 independent experiments . Schematic on right shows an overview of pathways leading to caspase - 8 activation .

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

AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (UT) or transfected with STAg as indicated for 18 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows an overview of pathways leading to caspase-8 activation.


Propidium iodide ( PI ) uptake from MDMs transfected with non - targeting control ( CTRL ) or GBP1 siRNA then left untreated ( UT ) , treated with LPS or transfection reagent ( Lipofectamine ) only or transfected with LPS for 4 h . under curve ( AUC ) from real - time assays plotted as mean ± from n = 4 independent experiments . Schematic on right shows an overview of pathways leading to caspase - 4 activation .

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

Propidium iodide (PI) uptake assay from MDMs transfected with non-targeting control (CTRL) or GBP1 siRNA and then left untreated (UT), treated with LPS or transfection reagent (Lipofectamine) only or transfected with LPS for 4 h. Area under the curve (AUC) from real-time assays plotted as mean ± SEM from n = 4 independent experiments. Schematic on right shows an overview of pathways leading to caspase-4 activation.


a , Diagram of cycle composed of G1 , S , G2 , M phases ( not to scale ) . Phase durations were quantified by time - lapse microscopy using a PCNA - mCherry reporter to identify four discrete events during lifetime of an were acquired every 10 min .

O O O O O O O O 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

a, Diagram of the cell cycle composed of G1, S, G2, and M phases (not to scale). Phase durations were quantified by time-lapse fluorescence microscopy using a PCNA-mCherry reporter to identify four discrete events during the lifetime of an individual cell Images were acquired every 10 min.


b , Mean phase durations in RPE , U2OS , H9 lines . Error bars represent standard deviations .

b, Mean phase durations in RPE, U2OS, and H9 cell lines. Error bars represent standard deviations.


c , Coefficient of variation ( CV ) of phase durations .

c, Coefficient of variation (CV) of phase durations.


d , of total variation in cycle duration contributed by phases .

d, Percentage of the total variation in cell cycle duration contributed by individual phases.


e , Correlations between cycle phase durations . Sample sizes were adequate to detect correlations n = 125 ( RPE ) , 130 ( U2OS ) , 113 ( H9 ) . R2 , square of Pearson correlation coefficient .

O O O O O O O O O O O O O O O O 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, Correlations between individual cell cycle phase durations. Sample sizes were adequate to detect correlations n = 125 (RPE), 130 (U2OS), 113 (H9). R2, square of Pearson correlation coefficient.


b , Shift in phase durations of RPE overexpressing Myc . boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : Myc , n =

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 B-CONTROLLED_VAR O O O

b, Shift in phase durations of RPE cells overexpressing Myc. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: Myc, n = 116


c , Pairwise correlation between cycle phase durations of RPE overexpressing Myc . Number of : Myc , n =

c, Pairwise correlation between cell cycle phase durations of RPE cells overexpressing Myc. Number of cells: Myc, n = 116


e , Shift in phase durations of RPE treated with 50 ng / mL aphidicolin . boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : aphidicolin , n =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Shift in phase durations of RPE cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 115


f , Pairwise correlation between cycle phase durations under aphidicolin treatment . Number of : aphidicolin , n =

f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 115


h , Shift in phase durations of RPE incubated at 34 ° C . boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : 34 ° C , n =

h, Shift in phase durations of RPE cells incubated at 34°C. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: 34°C, n = 122


i , Pairwise correlation between phase durations for incubated at 34 ° C . Number of : 34 ° C , n =

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

i, Pairwise correlation between phase durations for cells incubated at 34°C. Number of cells: 34°C, n = 122


k , Shift in phase durations of RPE treated with NCS . , boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * , P < 1 × 10 - 10 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : NCS , n = 119 .

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

k, Shift in phase durations of RPE cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 119.


l , Pairwise correlation between phase durations for treated with NCS . Number of : NCS , n = 119 .

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

l, Pairwise correlation between phase durations for cells treated with NCS. Number of cells: NCS, n = 119.


g , Shifts in phase durations for RPE treated with 2 μM CVT - 313 . A boxplot representing distributions of durations in untreated is underlaid for comparison . * , P < 1 × 10 - 5 , 2 - sided Kolmogorov - Smirnov . ( n = 117 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Shifts in phase durations for RPE cells treated with 2 μM CVT-313. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 117 cells).


h , Pairwise correlation between cycle phase durations upon treatment with CVT - 313 .

h, Pairwise correlation between cell cycle phase durations upon treatment with CVT-313.


j , Shifts in phase durations for RPE overexpressing cyclin D . A boxplot representing distributions of durations in untreated is underlaid for comparison . * , P < 1 × 10 - 5 , 2 - sided Kolmogorov - Smirnov . ( n = 113 ) .

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

j, Shifts in phase durations for RPE cells overexpressing cyclin D. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 113 cells).


k , Pairwise correlation between cycle phase durations upon overexpression of cyclin D . P indicates P - value .

k, Pairwise correlation between cell cycle phase durations upon overexpression of cyclin D. P indicates P-value.


b , Shift in phase durations of U2OS treated with NCS . , boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : NCS , n =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Shift in phase durations of U2OS cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 114


c , Pairwise correlation between phase durations for U2OS treated with NCS . Number of : NCS , n =

c, Pairwise correlation between phase durations for U2OS cells treated with NCS. Number of cells: NCS, n = 114


e , Shift in phase durations of U2OS treated with 50 ng / mL aphidicolin . boxplots representing distributions of phase durations in untreated are underlaid for comparison . * , P < 1 × 10 - 5 ; * * * , P < 1 × 10 - 20 , 2 - sided Kolmogorov - Smirnov . Number of : aphidicolin , n = 153 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Shift in phase durations of U2OS cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 153.


f , Pairwise correlation between cycle phase durations under aphidicolin treatment . Number of : aphidicolin , n = 153 .

f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 153.


B . Comparison of ΔPrP expression . Representative immunoblot is shown for detection of ( 3F4 ) MoPrP ΔPrPs in transiently transfected N2a using mAb 3F4 . ΔPrPs are similar in expression level glycosylation appearance , except for Δ159 - 167 which has an extra fragment larger than diglycoform ( lane 7 , arrow ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Comparison of ΔPrP expression levels. Representative immunoblot is shown for detection of (3F4)MoPrP and ΔPrPs in transiently transfected N2a cells using mAb 3F4. ΔPrPs are similar in expression level glycosylation appearance, except for Δ159-167 which has an extra fragment larger than the diglycoform (lane 7, arrow).


C . ΔPrPs are not converted into PK - resistant PrP in prion - infected . 22L - ScN2a were transiently transfected with ( 3F4 ) MoPrP , Δ159 or Δ159 - 175 , lysates digested with PK or not . Immunoblot was done using mAb 3F4 .

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

C. ΔPrPs are not converted into PK-resistant PrP in prion-infected cells. 22L-ScN2a cells were transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, and cell lysates digested with PK or not. Immunoblot was done using mAb 3F4.


. D . Pilot study for dominant - negative inhibition ( DNI ) of Δ159 Δ159 - 175 . DNI was assessed by co - transfecting 22L - ScN2a with ( 3F4 ) MoPrP Δ159 or Δ159 - 175 , respectively , testing for PK - resistant ( 3F4 ) MoPrP . Left panel shows representative immunoblot ( mAb 3F4 ) right panel statistical of quantified PK - res of a triplicate experiment . Empty pcDNA3 . 1 plasmid was used as control for co - transfection in lane 1 . error bars indicate standard deviation . * , p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Pilot study for dominant-negative inhibition (DNI) of Δ159 and Δ159-175. DNI was assessed by co-transfecting 22L-ScN2a cells with (3F4)MoPrP and Δ159 or Δ159-175, respectively, and testing for PK-resistant (3F4)MoPrP. Left panel shows representative immunoblot (mAb 3F4) and right panel the statistical analysis of quantified PK-res levels of a triplicate experiment. Empty pcDNA3.1 plasmid was used as control for co-transfection in lane 1. The error bars indicate standard deviation. *, p<0.05.


A . N - linked glycans of ΔPrPs are not - type . Immunoblot comparing EndoH - non - digested lysates of ΔPrP159 Δ159 - 175 transfected . Left panel was probed with mAb 3F4 mAb , detecting transiently expressed ΔPrPs . On right panel , was re - probed with mAb 4H11 , detecting total PrP . Arrowheads depict deglycosylated ΔPrPs .

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 B-CONTROLLED_VAR O O O O O O

A. N-linked glycans of ΔPrPs are not complex-type. Immunoblot comparing EndoH- and non-digested lysates of ΔPrP159 and Δ159-175 transfected cells. Left panel was probed with mAb 3F4 mAb, detecting transiently expressed ΔPrPs. On the right panel, the membrane was re-probed with mAb 4H11, detecting total PrP. Arrowheads depict deglycosylated ΔPrPs.


B . additional band of Δ159 - 167 represents a third N - glycan sensitive to EndoH . Upon EndoH digestion , bands converge into a non - glycoform band ( lane 3 4 ) . In Δ159 - 167 ( 169 ) third glycosylation site is not present anymore .

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

B. The additional band of Δ159-167 represents a third N-glycan sensitive to EndoH. Upon EndoH digestion, all bands converge into a non-glycoform band (lane 3 and 4). In Δ159-167(169) the third glycosylation site is not present anymore.


C . ΔPrPs are mainly distributed in intracellular compartments rather than on surface . Confocal microscopy of N2a transiently transfected with ( 3F4 ) MoPrP , Δ159 or Δ159 - 175 , either permeabilized ( right ) or non - permeabilized ( left ) with 0 . 2 % TX100 after fixation . were immuno - labeled with mAb 3F4 . ( 3F4 ) MoPrP - expressing show bright immunopositivity on surface , whereas ΔPrPs are not observed on surface . Also intracellular differs strongly between ( 3F4 ) MoPrP ΔPrP expressing Scale bars , 25 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ΔPrPs are mainly distributed in intracellular compartments rather than on the cell surface. Confocal microscopy images of N2a cells transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, either permeabilized (right) or non-permeabilized (left) with 0.2% TX100 after fixation. Cells were immuno-labeled with mAb 3F4. (3F4)MoPrP-expressing cells show bright immunopositivity on the cell surface, whereas ΔPrPs are not observed on the cell surface. Also intracellular staining differs strongly between (3F4)MoPrP and ΔPrP expressing cells Scale bars, 25 µm.


A . ΔPrPs are insoluble in detergent solution . Immunoblots showing partition of Δ159 Δ159 - 175 in supernatant ( Sup ) pellet fraction after ultracentrifugation of lysates supplemented with 4 % sarcosyl . Upper panel is mAb 3F4 , lower panel is re - probed with mAb 4H11 detecting also endogenous wild - type PrP besides ΔPrPs . majority of ΔPrPs is found in insoluble fraction in both infected non - infected ( upper panel , lanes 3 , 4 , 7 8 ) . Wild - type PrP almost completely partitions into soluble fraction ( Sup ) in non - infected N2a with present conditions . PrP in pellet fractions of non - infected N2a is from ΔPrPs as can be seen from banding pattern ( Lane 7 8 , square bracket ) . PrP in lanes 3 4 ( lower panel ) is PrP27 - 30 .

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

A. ΔPrPs are insoluble in detergent solution. Immunoblots showing partition of Δ159 and Δ159-175 in supernatant (Sup) and pellet fraction after ultracentrifugation of lysates supplemented with 4% sarcosyl. Upper panel is mAb 3F4, lower panel is membrane re-probed with mAb 4H11 detecting also endogenous wild-type PrP besides ΔPrPs. The majority of ΔPrPs is found in the insoluble fraction in both infected and non-infected cells (upper panel, lanes 3, 4, 7 and 8). Wild-type PrP almost completely partitions into the soluble fraction (Sup) in non-infected N2a cells with present conditions. PrP in pellet fractions of non-infected N2a cells is from ΔPrPs as can be seen from the banding pattern (Lane 7 and 8, square bracket). PrP in lanes 3 and 4 (lower panel) is PrP27-30.


B . ΔPrPs are moderately resistant to chymotrypsin digestion . Immunoblots showing chymotrypsin - resistant fragments of Δ159 Δ159 - 175 from transiently transfected N2a lysates digested with indicated concentrations of chymotrypsin ( Chy ) . Note that full - diglycoform of ΔPrP still remains at 8 µg / ml Chy ( lanes 5 10 , upper panel , closed arrowhead ) , whereas that of endogenous wild - type PrP is completely digested ( lanes 5 10 , lower panel , open arrowhead ) only small amounts of truncated fragments remained ( curly bracket ) . Other smaller fragments in lane 5 10 ( square - brackets ) represent ΔPrPs .

O O O O O O O B-CONTROLLED_VAR 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 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-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

B. ΔPrPs are moderately resistant to chymotrypsin digestion. Immunoblots showing chymotrypsin-resistant fragments of Δ159 and Δ159-175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin (Chy). Note that full-length diglycoform of ΔPrP still remains at 8 µg/ml Chy (lanes 5 and 10, upper panel, closed arrowhead), whereas that of endogenous wild-type PrP is completely digested (lanes 5 and 10, lower panel, open arrowhead) and only small amounts of truncated fragments remained (curly bracket). Other smaller fragments in lane 5 and 10 (square-brackets) represent ΔPrPs.


A . ΔPrPs are substantially soluble in detergent without GdnHCl denaturation . Immunoblots probed with mAbs 3F4 ( left ) or 4H11 ( right , re - probed ) showing ΔPrPs endogenous PrP in detergent aqueous phases of TX114 lysates prepared from N2a transfected with Δ159 or Δ159 - 175 . Det denotes detergent phase , Aq aqueous phase .

O O O O O O O O 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

A. ΔPrPs are substantially soluble in detergent without GdnHCl denaturation. Immunoblots probed with mAbs 3F4 (left) or 4H11 (right, membrane re-probed) showing ΔPrPs and endogenous PrP in detergent and aqueous phases of TX114 lysates prepared from N2a cells transfected with Δ159 or Δ159-175. Det denotes detergent phase, Aq aqueous phase.


B . ΔPrPs are highly hydrophobic after denaturation with GdnHCl . Representative immunoblot of TX114 - treated lysates after denaturation with 3 M GdnHCl developed with mAb 3F4 showing Δ159 Δ159 - 175 almost exclusively in detergent phase ( lanes 1 - 4 ) . PIPLC treatment ( + , even lanes ) was done after denaturation with GdnHCl or not ( − , uneven lanes ) . PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions .

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

B. ΔPrPs are highly hydrophobic after denaturation with GdnHCl. Representative immunoblot of TX114-treated lysates after denaturation with 3 M GdnHCl and developed with mAb 3F4 showing Δ159 and Δ159-175 almost exclusively in detergent phase (lanes 1-4). PIPLC treatment (+, even lanes) was done after denaturation with GdnHCl or not (−, uneven lanes). PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions.


C . Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion . After further diluting out GdnHCl by phase - separation removal of aqueous phase containing GdnHCl , PIPLC was able to digest ΔPrPs . Left two panels ( upper mAb 3F4 , lower 4H11 ) are shorter expositions , right panels longer expositions to show also difference in PrP in detergent phase . Under these conditions a substantial reduction of ΔPrPs in detergent phase was achieved by PIPLC digestion ( e . g . upper - left panel , lanes 1 , 3 vs . 2 , 4 ) . Migration of ΔPrPs in aqueous phase of PIPLC - digested samples was slightly slower than that of PIPLC - undigested samples ( − ) ( upper - right panel , lane 9 vs . 10 ) .

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 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 B-MEASURED_VAR 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 B-CONTROLLED_VAR 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

C. Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion. After further diluting out GdnHCl by phase-separation and removal of the aqueous phase containing GdnHCl, PIPLC was able to digest ΔPrPs. Left two panels (upper mAb 3F4, lower 4H11) are shorter expositions, right panels longer expositions to show also difference in PrP levels in the detergent phase. Under these conditions a substantial reduction of ΔPrPs in the detergent phase was achieved by PIPLC digestion (e.g. upper-left panel, lanes 1, 3 vs. 2, 4). Migration of ΔPrPs in the aqueous phase of PIPLC-digested samples was slightly slower than that of PIPLC-undigested samples (−) (upper-right panel, lane 9 vs. 10).


A . Degradation of ΔPrPs by lysosomal proteasomal systems . ( Left ) N2a transiently transfected with Δ159 or Δ159 - 175 were treated for 7 hours with inhibitors either for lysosomal degradation ( bafilomycin A1 , Baf , 120 nM ) , autophagy ( 3 - methyladenine , 3MA , 10 mM ) , or proteasome ( MG132 , 10 µM ) lysates analyzed in immunoblot for expression of Δ159 Δ159 - 175 ( mAb 3F4 ) . Square bracket bracket denote diglycoforms nonglycoforms of ΔPrPs , respectively . An increment in diglycoforms is observed in Baf - 3MA - treated , whereas in MG132 - treated also non - glycoforms are increased . ( Right ) A graph showing of PrP bands by densitometric . Data from 3 independent ( one in duplicate ) experiments for Δ159 2 independent ( one in duplicate ) for Δ159 - 175 were statistically analyzed for mean standard deviation ( error 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

A. Degradation of ΔPrPs by lysosomal and proteasomal systems. (Left) N2a cells transiently transfected with Δ159 or Δ159-175 were treated for 7 hours with inhibitors either for lysosomal degradation (bafilomycin A1, Baf, 120 nM), autophagy (3-methyladenine, 3MA, 10 mM), or the proteasome (MG132, 10 µM) and cell lysates analyzed in immunoblot for expression of Δ159 and Δ159-175 (mAb 3F4). Square bracket and bracket denote diglycoforms and nonglycoforms of ΔPrPs, respectively. An increment in diglycoforms is observed in Baf- and 3MA-treated cells, whereas in MG132-treated cells also the non-glycoforms are increased. (Right) A graph showing quantification of all PrP bands by densitometric analysis. Data from 3 independent (one in duplicate) experiments for Δ159 and 2 independent (one in duplicate) for Δ159-175 were statistically analyzed for mean and standard deviation (error bars).


B . ΔPrPs can be localized in LAMP1 - immunopositive vesicles . Immunofluorescence shows distribution of ΔPrP ( mAb 3F4 ) LAMP1 in with or without 6M GdnHCl antigen - retrieval treatment . N2a transfected with ΔPrP159 were fixed , permeabilized incubated without ( upper panel ) or with 6M GdnHCl ( lower panel ) . With GndHCl treatment ΔPrP is diffusely distributed ( arrowheads ) co - localizes poorly with LAMP1 . After GdnHCl treatment ( lower panel ) , bright 3F4 - immunopositive ( arrows ) are observed which partly co - localize with LAMP1 - positive structures ( merge ) . Scale bars , 10 µm .

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

B. ΔPrPs can be localized in LAMP1-immunopositive vesicles. Immunofluorescence analysis shows distribution of ΔPrP (mAb 3F4) and LAMP1 in cells with or without 6M GdnHCl antigen-retrieval treatment. N2a cells transfected with ΔPrP159 were fixed, permeabilized and incubated without (upper panel) or with 6M GdnHCl (lower panel). With GndHCl treatment ΔPrP is diffusely distributed (arrowheads) and co-localizes poorly with LAMP1. After GdnHCl treatment (lower panel), bright 3F4-immunopositive puncta (arrows) are observed which partly co-localize with LAMP1-positive structures (merge). Scale bars, 10 µm.


A . DNI efficiencies of ΔPrPs are inversely correlated to deletion . Representative immunoblot showing PK - resistant PrPSc moiety of ( 3F4 ) MoPrP co - expressed with indicated ΔPrPs in 22L - ScN2a . Quantitative is shown below as scheme . Relative PrPSc were calculated as proportion of PK - resistant ( 3F4 ) MoPrP co - transfected with each ΔPrP to that of co - transfected with empty vector control in same experiment . Data from 4 ( for Δ159 - 165 , Δ159 - 167 Δ159 - 167 ( 169 ) only 3 ) independent experiments were statistically analyzed for mean standard deviation ( error bars ) .

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

A. DNI efficiencies of ΔPrPs are inversely correlated to deletion size. Representative immunoblot showing PK-resistant PrPSc moiety of (3F4)MoPrP co-expressed with indicated ΔPrPs in 22L-ScN2a cells. Quantitative analysis is shown below as scheme. Relative PrPSc levels were calculated as proportion of PK-resistant (3F4)MoPrP co-transfected with each ΔPrP to that of co-transfected with empty vector control in the same experiment. Data from 4 (for Δ159-165, Δ159-167 and Δ159-167(169) only 3) independent experiments were statistically analyzed for mean and standard deviation (error bars).


B . DNI of ΔPrP with a deletion in C - terminal part of H1 ∼ H2 . Δ171 - 175 ( see Fig . 7A ) was transiently expressed in N2a ( left panel ) DNI efficiency evaluated in 22L - ScN2a ( right panel ) ( mAb 3F4 ) . Despite much shorter deletion , Δ171 - 175 exerted only inefficient DNI , similar to Δ159 - 175 ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DNI of ΔPrP with a deletion in the C-terminal part of H1∼H2. Δ171-175 (see Fig. 7A) was transiently expressed in N2a cells (left panel) and DNI efficiency evaluated in 22L-ScN2a cells (right panel) (mAb 3F4). Despite the much shorter deletion size, Δ171-175 exerted only inefficient DNI, similar to Δ159-175 (right panel).


. B . Δ31 - 160 variants are expressed at comparable have EndoH - sensitive - resistant N - glycans . Immunoblot comparing EndoH - digested non - digested samples from N2a transiently transfected with Δ31 - 160 variants , probed with anti - PrP mAb C16 - S raised against C - terminal portion of H3 . Non - Tf , samples prepared from N2a without transfection . Arrowhead denotes deglycosylated Δ159 ( lane 11 ) , bracket deglycosylated fragments , square bracket EndoH - resistant fragments . Asterisks indicate non - specific bands . Δ31 - 160 variants are not completely deglycosylated by EndoH , resulting in EndoH - resistant fragments ( square bracket ) which are observed also in reducing conditions ( right panel ; + / − dithiothreitol ( DTT ) treatment ) . Note that endogenous wild - type PrPc was not detected under used conditions .

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

. B. Δ31-160 variants are expressed at comparable levels and have EndoH-sensitive and -resistant N-glycans. Immunoblot comparing EndoH-digested and non-digested samples from N2a cells transiently transfected with Δ31-160 variants, probed with anti-PrP mAb C16-S raised against the C-terminal portion of H3. Non-Tf, samples prepared from N2a cells without transfection. Arrowhead denotes deglycosylated Δ159 (lane 11), bracket deglycosylated fragments, square bracket EndoH-resistant fragments. Asterisks indicate non-specific bands. Δ31-160 variants are not completely deglycosylated by EndoH, resulting in EndoH-resistant fragments (square bracket) which are observed also in reducing conditions (right panel; +/− dithiothreitol (DTT) treatment). Note that endogenous wild-type PrPc was not detected under used conditions.


C . DNI of Δ31 - 160 variants is similarly inversely related to of deletion dependent on intact C - terminal H1 - H2 portion . Immunoblot probed with mAb 3F4 showing PK - resistant ( 3F4 ) MoPrP co - transfected with indicated constructs into 22L - ScN2a ( left panel ) . Δ31 - 160 was slightly more effective than Δ159 . Right panel shows of results as obtained from a triplicate experiment , using densitometry on ImageJ . Bars illustrate mean ± standard deviation . * , p < 0 . 05 .

O O O O O O O O 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

C. DNI of Δ31-160 variants is similarly inversely related to size of deletion and dependent on intact C-terminal H1-H2 portion. Immunoblot probed with mAb 3F4 showing PK-resistant (3F4)MoPrP co-transfected with indicated constructs into 22L-ScN2a cells (left panel). Δ31-160 was slightly more effective than Δ159. Right panel shows quantification of results as obtained from a triplicate experiment, using densitometry on ImageJ. Bars illustrate mean ± standard deviation. *, p<0.05.


D . Δ31 - 160 forms spontaneous with moderate chymotrypsin ( Chy ) resistance . Immunoblots probed with mAbs C16 - S ( upper panel ) or 3F4 ( lower panel ) for comparing Chy - resistant fragments of Δ31 - 160 with that of Δ159 . Lysates from N2a expressing Δ159 or Δ31 - 160 were digested with indicated concentrations of Chy for 30 minutes then digested with PNGaseF . A sample from non - transfected N2a is shown in lane 5 . Asterisks indicate non - specific C16 - S bands . Arrowhead denotes deglycoform of Δ159 , bracket deglycoform of Δ31 - 160 .

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

D. Δ31-160 forms spontaneous aggregates with moderate chymotrypsin (Chy) resistance. Immunoblots probed with mAbs C16-S (upper panel) or 3F4 (lower panel) for comparing Chy-resistant fragments of Δ31-160 with that of Δ159. Lysates from N2a cells expressing Δ159 or Δ31-160 were digested with indicated concentrations of Chy for 30 minutes and then digested with PNGaseF. A sample from non-transfected N2a cells is shown in the lane 5. Asterisks indicate non-specific C16-S bands. Arrowhead denotes deglycoform of Δ159, bracket deglycoform of Δ31-160.


B ( Top ) Semiquantitative RT - PCR of UPF1SL or UPF1LL transcript following transfection of HEK - 293 with a NT siRNA or a siRNA that specifically targets UPF1LL isoform . ( Bottom ) Venn diagram ( to scale ) of overlapping targets identified from - seq following total UPF1 or UPF1LL - specific knockdown . Depicted are genes that increased in abundance at least 1 . 4 - fold ( FDR < 0 . 05 ) met read cutoffs in both datasets . P value indicates enrichment of genes that increased in abundance at least 1 . 4 - fold ( FDR < 0 . 05 ) with UPF1LL - specific knockdown among those regulated by total UPF1 , as determined by Fisher ' s exact .

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

B (Top) Semiquantitative RT-PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK-293 cells with a NT siRNA or a siRNA that specifically targets the UPF1LL isoform. (Bottom) Venn diagram (to scale) of overlapping targets identified from RNA-seq following total UPF1 or UPF1LL-specific knockdown. Depicted are genes that increased in abundance at least 1.4-fold (FDR < 0.05) and met read count cutoffs in both datasets. P value indicates enrichment of genes that increased in abundance at least 1.4-fold (FDR < 0.05) with UPF1LL-specific knockdown among those regulated by total UPF1, as determined by Fisher's exact test.


C Heat map of changes in relative mRNA abundance for genes encoding NMD factors , as determined from - seq following transfection of HEK - 293 with a siRNA that targets both UPF1 isoforms ( UPF1total ) or a siRNA that specifically targets UPF1LL isoform .

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

C Heat map of changes in relative mRNA abundance for genes encoding NMD factors, as determined from RNA-seq following transfection of HEK-293 cells with a siRNA that targets both UPF1 isoforms (UPF1total) or a siRNA that specifically targets the UPF1LL isoform.


D Density plot of changes in relative mRNA abundance as determined by - seq in SMG7ko / SMG6kd , relative to a parental treated with control siRNAs Genes were categorized as up - regulated by siUPF1total only , siUPF1LL only , or both siUPF1total siUPF1LL . Statistical significance was determined by K - W , with Dunn ' s correction for multiple comparisons .

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

D Density plot of changes in relative mRNA abundance as determined by RNA-seq in SMG7ko/SMG6kd cells, relative to a parental cell line treated with control siRNAs Genes were categorized as up-regulated by siUPF1total only, siUPF1LL only, or both siUPF1total and siUPF1LL. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.


E RT - qPCR of indicated transcripts following transfection of HEK - 293 with indicated siRNAs . Relative fold changes are in reference to NT siRNA . Asterisk ( * ) indicates P < 0 . 05 , as determined by two - way ANOVA . Black dots represent data points error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed indicates log2 ( fold change ) of + 0 . 5 . PTC + indicates use of primers specific to transcript isoforms with validated poison

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with the indicated siRNAs. Relative fold changes are in reference to NT siRNA. Asterisk (*) indicates P < 0.05, as determined by two-way ANOVA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed line indicates log2(fold change) of +0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


F Gene ontology of 1621 genes that increased in expression at least 1 . 4 - fold upon UPF1LL - specific knockdown in HEK - 293 under normal conditions . Genes may map to multiple categories .

O O O O O O O O O O O O O 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 Gene ontology analysis of 1621 genes that increased in expression at least 1.4-fold upon UPF1LL-specific knockdown in HEK-293 cells under normal cellular conditions. Genes may map to multiple categories.


B Scatterplot of CLIP - UPF1LL vs CLIP - UPF1SL RIP - seq efficiency . mRNAs were binned according to 3 ' UTR ( short , medium , or long ) .

O O O O O B-MEASURED_VAR 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 Scatterplot of CLIP-UPF1LL vs CLIP-UPF1SL RIP-seq efficiency. mRNAs were binned according to 3'UTR length (short, medium, or long).


C Density plot of recovered mRNAs in CLIP - UPF1LL or CLIP - UPF1SL affinity purifications . mRNAs were binned according to 3 ' UTR . Statistical significance was determined by K - S .

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

C Density plot of recovered mRNAs in CLIP-UPF1LL or CLIP-UPF1SL affinity purifications. mRNAs were binned according to 3'UTR length. Statistical significance was determined by K-S test.


D Density plot of recovered mRNAs in CLIP - UPF1LL affinity purifications relative to that of CLIP - UPF1SL . mRNAs were subdivided by PTBP1 / or hnRNP L motif density within 3 ' UTR , as indicated by gradient triangle . Statistical significance was determined by K - W , with Dunn ' s correction for multiple comparisons .

O O 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

D Density plot of recovered mRNAs in CLIP-UPF1LL affinity purifications relative to that of CLIP-UPF1SL. mRNAs were subdivided by PTBP1 and/or hnRNP L motif density within the 3'UTR, as indicated by the gradient triangle. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.


E RT - qPCR of indicated transcripts from CLIP - UPF1 overexpression - seq experiments . Relative fold changes are in reference to GFP expressing control . Significance of CLIP - UPF1SL or CLIP - UPF1LL overexpression was compared to GFP expressing control . Asterisk ( * ) indicates P < 0 . 05 , as determined by two - way ANOVA . Black dots represent data points error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed lines indicate log2 ( fold change ) of ± 0 . 5 . For protected mRNAs , motif density of PTBP1 / hnRNP L within 3 ' UTR is indicated . PTC + indicates use of primers specific to transcript isoforms with validated poison

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 B-CONTROLLED_VAR 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

E RT-qPCR analysis of indicated transcripts from CLIP-UPF1 overexpression RNA-seq experiments. Relative fold changes are in reference to the GFP expressing control line. Significance of CLIP-UPF1SL or CLIP-UPF1LL overexpression was compared to the GFP expressing control line. Asterisk (*) indicates P < 0.05, as determined by two-way ANOVA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. For protected mRNAs, the motif density of PTBP1/hnRNP L within the 3'UTR is indicated. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


A Semiquantitative RT - PCR of UPF1SL or UPF1LL transcript following transfection of HEK - 293 with indicated siRNAs .

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

A Semiquantitative RT-PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK-293 cells with the indicated siRNAs.


B RT - qPCR of indicated transcripts following transfection of a NT siRNA or SRSF1 - specific siRNA under conditions of CLIP - UPF1SL or CLIP - UPF1LL overexpression . Relative fold changes are in reference to GFP expressing control treated with a NT siRNA . Asterisk ( * ) indicates P < 0 . 05 , as determined by unpaired Student ' s t - . Black dots represent data points error bars indicate mean ± SD ( n = 3 biological replicates ) .

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 B-CONTROLLED_VAR 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

B RT-qPCR analysis of indicated transcripts following transfection of a NT siRNA or SRSF1-specific siRNA under conditions of CLIP-UPF1SL or CLIP-UPF1LL overexpression. Relative fold changes are in reference to the GFP expressing control line treated with a NT siRNA. Asterisk (*) indicates P < 0.05, as determined by unpaired Student's t-test. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates).


C Density plot of changes in relative mRNA abundance as determined by - seq following SRSF1 overexpression Genes were categorized as up - regulated by siUPF1total only , siUPF1LL only , or both siUPF1total siUPF1LL . Statistical significance was determined by K - W , with Dunn ' s correction for multiple comparisons .

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

C Density plot of changes in relative mRNA abundance as determined by RNA-seq following SRSF1 overexpression Genes were categorized as up-regulated by siUPF1total only, siUPF1LL only, or both siUPF1total and siUPF1LL. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.


D Density plot as in ( C ) , with genes binned according to enrichment in CLIP - UPF1LL or CLIP - UPF1SL affinity purifications .

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

D Density plot as in (C), with genes binned according to enrichment in the CLIP-UPF1LL or CLIP-UPF1SL affinity purifications.