( H ) Boxplots compare the peak intensities inside individual vDNA foci for MOIs 10 , 50 and 100 . Central lines in boxes define medians , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . Intensities increase significantly with MOI ( Wilcoxon tests : MOI 10 vs . MOI 50 : * * * p ≈ 5 ⋅ 10 − 16 ; MOI 50 vs . MOI 100 : * * * p ≈ 5 ⋅ 10 − 5 ; MOI 10 vs . MOI 100 : * * * p ≈ 2 ⋅ 10 − 23 ) . Data from one experiment . A replicate showed similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots compare the peak intensities inside individual vDNA foci for MOIs 10, 50 and 100. Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Intensities increase significantly with MOI (Wilcoxon tests: MOI 10 vs. MOI 50: *** p ≈ 5 ⋅ 10 − 16; MOI 50 vs. MOI 100: ***p ≈ 5 ⋅ 10 − 5; MOI 10 vs. MOI 100: ***p ≈ 2 ⋅ 10 − 23). Data from one experiment. A replicate showed similar results.


( I ) Histograms show the number of foci per nucleus for MOIs of 10 ( blue ) , 50 ( green ) and 100 ( red ) .

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

(I) Histograms show the number of foci per nucleus for MOIs of 10 (blue), 50 (green) and 100 (red).


vDNA foci are also foci of vRNA . ( A ) Dual - color image of an infected ThP1 cell showing the viral DNA ( red ) and the RNA visualized by RNA - FISH ( green ) . Arrows show foci containing both vDNA and vRNA . The bottom row shows the boxed region magnified , with vDNA and vRNA displayed separately and in combination . See also Appendix Fig . S6 .

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

vDNA foci are also foci of vRNA. (A) Dual-color image of an infected ThP1 cell showing the viral DNA (red) and the RNA visualized by RNA-FISH (green). Arrows show foci containing both vDNA and vRNA. The bottom row shows the boxed region magnified, with vDNA and vRNA displayed separately and in combination. See also Appendix Fig. S6.


( C ) Scatter plot shows the intensities of vDNA and vRNA in colocalizing foci with the Spearman correlation r and associated p - value .

O O 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) Scatter plot shows the intensities of vDNA and vRNA in colocalizing foci with the Spearman correlation r and associated p-value.


In ThP1 cells co - infected with a HIV1 - GFP virus and a HIV1 - LUC virus , nuclear foci contain mixtures of both viruses . ( D ) Dual - color image shows RNA - FISH against GFP ( red ) and RNA - FISH against LUC ( green ) . Arrows show foci containing both GFP vRNA and LUC vRNA . The bottom row shows the boxed region magnified , with GFP vRNA and LUC vRNA displayed separately and in combination . See Appendix Fig . S7 .

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

In ThP1 cells co-infected with a HIV1-GFP virus and a HIV1-LUC virus, nuclear foci contain mixtures of both viruses. (D) Dual-color image shows RNA-FISH against GFP (red) and RNA-FISH against LUC (green). Arrows show foci containing both GFP vRNA and LUC vRNA. The bottom row shows the boxed region magnified, with GFP vRNA and LUC vRNA displayed separately and in combination. See Appendix Fig. S7.


( F ) Scatter plot shows intensities of GFP RNA and LUC RNA in colocalizing foci with the Spearman correlation r and associated p - value .

O O O 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) Scatter plot shows intensities of GFP RNA and LUC RNA in colocalizing foci with the Spearman correlation r and associated p-value.


Images of infected ( right ) or uninfected ( left ) ThP1 cells showing the vDNA ( EdU ) in red , the nucleus ( DAPI ) in blue , and selected nuclear body factors in green . ( A ) Green image shows immunolabeling of CPSF6 .

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

Images of infected (right) or uninfected (left) ThP1 cells showing the vDNA (EdU) in red, the nucleus (DAPI) in blue, and selected nuclear body factors in green. (A) Green image shows immunolabeling of CPSF6.


( B ) Green image shows RNA - FISH against NEAT1 .

(B) Green image shows RNA-FISH against NEAT1.


( C ) Green image shows immunolabeling of SC35 . See also Appendix Figs . S9 - 14 .

(C) Green image shows immunolabeling of SC35. See also Appendix Figs. S9-14.


( D ) Boxplots show Pearson correlations r between vDNA ( EdU ) and CPSF6 , NEAT1 or SC35 in 17 - 19 regions of interest ( ROIs ) . Central lines in boxes define medians , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . Significance of positive or negative correlations was assessed using the Costes method of random ROI shifts . Highly significant ( p < 0 . 01 ) positive correlations between vDNA and CPSF6 intensities are found in 16 out of 19 ROIs ; highly significant negative correlations between vDNA and NEAT1 are found in 19 / 19 ROIs , and highly significant positive correlations between vDNA and SC35 are found for 16 / 17 ROIs . Data are from one experiment . For CPSF6 , the experiment was repeated with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots show Pearson correlations r between vDNA (EdU) and CPSF6, NEAT1 or SC35 in 17-19 regions of interest (ROIs). Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Significance of positive or negative correlations was assessed using the Costes method of random ROI shifts. Highly significant (p<0.01) positive correlations between vDNA and CPSF6 intensities are found in 16 out of 19 ROIs; highly significant negative correlations between vDNA and NEAT1 are found in 19/19 ROIs, and highly significant positive correlations between vDNA and SC35 are found for 16/17 ROIs. Data are from one experiment. For CPSF6, the experiment was repeated with similar results.


( E ) Intensity profiles of EdU and DAPI along the dotted lines in the vDNA images above .

(E) Intensity profiles of EdU and DAPI along the dotted lines in the vDNA images above.


( A ) Image of a ThP1 cell infected with an integration - deficient HIV - 1 carrying the mutation D116A in the catalytic site of IN . The EdU - labeled vDNA is shown in red and the vRNA detected by RNA - FISH in green . The nucleus ( DAPI ) is shown in blue . See Appendix Fig . S12 for a larger image region .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Image of a ThP1 cell infected with an integration-deficient HIV-1 carrying the mutation D116A in the catalytic site of IN. The EdU-labeled vDNA is shown in red and the vRNA detected by RNA-FISH in green. The nucleus (DAPI) is shown in blue. See Appendix Fig. S12 for a larger image region.


( C ) Scatter plot shows vDNA and vRNA intensities in colocalizing vDNA and vRNA clusters with the Spearman correlation r and associated p - value .

(C) Scatter plot shows vDNA and vRNA intensities in colocalizing vDNA and vRNA clusters with the Spearman correlation r and associated p-value.


( D ) ThP1 cells were infected with HIV - 1 at an MOI of 20 , in absence ( left ) or presence ( right ) of nevirapine ( NVP , 10 µM ) . Blue : DAPI staining . Green : vRNA ( RNA - FISH ) . Red : vDNA ( EdU ) . ( E ) Scatter plots show intensities of vDNA and vRNA in detected vRNA clusters .

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

(D) ThP1 cells were infected with HIV-1 at an MOI of 20, in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Red: vDNA (EdU). (E) Scatter plots show intensities of vDNA and vRNA in detected vRNA clusters.


( F ) Image of a ThP1 cell infected by HIV - 1 with EU labeled RNA in absence ( left ) or presence ( right ) of nevirapine ( NVP , 10 µM ) . Blue : DAPI staining . Green : vRNA ( RNA - FISH ) . Grey : EU - labeled vRNA . See Appendix Fig . S19 for a larger image region . ( G ) Intensity profiles of EU and RNA - FISH along the dotted lines in panel F ( top ) .

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

(F) Image of a ThP1 cell infected by HIV-1 with EU labeled RNA in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Grey: EU-labeled vRNA. See Appendix Fig. S19 for a larger image region. (G) Intensity profiles of EU and RNA-FISH along the dotted lines in panel F (top).


The left panel shows the timeline of drug exposure experiments . ( A - D ) Images show vRNA and vDNA in an infected ThP1 cell ( MOI 20 ) for each of four experimental conditions . See Appendix Fig . S15 - S17 for images of larger regions . ( A , B ) ThP1 cells were exposed to NVP for 48 h ( A ) or 72 h after infection ( B ) , then NVP was washed out , and cells were fixed for click chemistry 24 h later . ( C ) Cells were exposed to PF74 for 72 h after infection , then fixed for click chemistry . ( D ) Cells were exposed to NVP for 24 h after infection , then NVP was washed out , and cells were exposed to PF74 , before being fixed for click chemistry 24 h later . ( E , F , H ) Scatter plots show vDNA and vRNA intensities in colocalizing clusters with Spearman correlation r and associated p - values . Replicates of the four experiments yielded similar results . ( G ) qPCR measures DNA synthesis ( left ) and nuclear import ( right ) at 72 h p . i . in absence of drug treatment , or after exposure to NVP or PF74 . Bars and error bars define mean and standard deviation , respectively . Samples were analyzed in triplicate and two biological replicates were performed .

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

The left panel shows the timeline of drug exposure experiments. (A-D) Images show vRNA and vDNA in an infected ThP1 cell (MOI 20) for each of four experimental conditions. See Appendix Fig. S15-S17 for images of larger regions. (A,B) ThP1 cells were exposed to NVP for 48 h (A) or 72 h after infection (B), then NVP was washed out, and cells were fixed for click chemistry 24 h later. (C) Cells were exposed to PF74 for 72 h after infection, then fixed for click chemistry. (D) Cells were exposed to NVP for 24 h after infection, then NVP was washed out, and cells were exposed to PF74, before being fixed for click chemistry 24 h later. (E,F,H) Scatter plots show vDNA and vRNA intensities in colocalizing clusters with Spearman correlation r and associated p-values. Replicates of the four experiments yielded similar results. (G) qPCR measures DNA synthesis (left) and nuclear import (right) at 72 h p.i. in absence of drug treatment, or after exposure to NVP or PF74. Bars and error bars define mean and standard deviation, respectively. Samples were analyzed in triplicate and two biological replicates were performed.


The left panel shows the timeline of infection and / or drug exposure experiments . ( A - D ) Percentage of GFP positive cells at 3 and 7 days post - infection analyzed by FACS in absence of EdU . ( A ) Uninfected control cells . ( B ) Untreated , infected ThP1 cells fixed at 3 d ( 72 h ) p . i . ( C ) Infected cells were exposed to NVP for 72 h , then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p . i . ( D ) Infected cells were cultured for 72 h p . i . , then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p . i . ( E - H ) Multicolor images of ThP1 cells infected with a GFP - reporter virus . vRNA ( RNA - FISH ) is labeled in red , GFP in green , and nuclei ( DAPI ) in blue . ( E ) Uninfected control cells . ( F ) Untreated , infected ThP1 cells fixed at 3 d ( 72 h ) p . i . ( G ) Infected cells were exposed to NVP for 72 h , then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p . i . ( H ) Infected cells were cultured for 72 h p . i . , then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p . i . The experiment was repeated three times .

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

The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Percentage of GFP positive cells at 3 and 7 days post-infection analyzed by FACS in absence of EdU. (A) Uninfected control cells. (B) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (C) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (D) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. (E-H) Multicolor images of ThP1 cells infected with a GFP-reporter virus. vRNA (RNA-FISH) is labeled in red, GFP in green, and nuclei (DAPI) in blue. (E) Uninfected control cells. (F) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (G) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (H) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. The experiment was repeated three times.


The left panel shows the timeline of infection and / or drug exposure experiments . ( A - D ) Multicolor images of vDNA , vRNA , DAPI and / or GFP in monocyte derived macrophages ( MDMs ) from two different donors , infected with HIV1 . Cells from donor 1 were infected with VSV - G - pseudotyped HIV - 1ΔEnv Vpx carrying a GFP reporter ( A ) and cells from donor 2 were infected with a VSV - G - pseudotyped HIV - 1ΔEnv without Vpx ( B - D ) . ( A , B ) Infected cells were left untreated and fixed at 6 d p . i . ( C ) Infected cells were treated with 10 µM Nevirapine ( NVP ) throughout the experiment and fixed at 6 d p . i . ( D ) Infected cells were exposed to NVP for 3 d p . i . , then NVP was washed out and cells were cultured for another 4 d and fixed at 7 d p . i . Arrows in ( A - D ) show the position of selected vRNA foci . ( E ) Boxplot shows the size of DNA foci in untreated infected cells from donor 2 imaged at 7 d p . i . ( B ) , measured by the FWHM of n = 24 foci . The red line defines the median , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points . ( F ) Boxplot shows Pearson ' s r , measuring colocalization of DNA with RNA foci in the same cells ( n = 20 ) . Center line defines the median , top and bottom limits define upper and lower quartiles , respectively . Whiskers show the full data range , except for outliers . Grey dots are individual data points .

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

The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Multicolor images of vDNA, vRNA, DAPI and/or GFP in monocyte derived macrophages (MDMs) from two different donors, infected with HIV1. Cells from donor 1 were infected with VSV-G-pseudotyped HIV-1ΔEnv Vpx carrying a GFP reporter (A) and cells from donor 2 were infected with a VSV-G-pseudotyped HIV-1ΔEnv without Vpx (B-D). (A,B) Infected cells were left untreated and fixed at 6 d p.i. (C) Infected cells were treated with 10 µM Nevirapine (NVP) throughout the experiment and fixed at 6 d p.i. (D) Infected cells were exposed to NVP for 3 d p.i., then NVP was washed out and cells were cultured for another 4 d and fixed at 7 d p.i. Arrows in (A-D) show the position of selected vRNA foci. (E) Boxplot shows the size of DNA foci in untreated infected cells from donor 2 imaged at 7 d p.i. (B), measured by the FWHM of n=24 foci. The red line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. (F) Boxplot shows Pearson's r, measuring colocalization of DNA with RNA foci in the same cells (n=20). Center line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points.


( G ) FACS analysis of Gag positive cells from donor 2 in different conditions . Red : uninfected cells . Blue : untreated infected cells at 7 d p . i . as in B . Orange : cells were treated with NVP for 7 d as in C . Green : infected cells were exposed to NVP for 3 d , then NVP was washed out and cells were cultured for another 4 d as in D . Median of fluorescence intensities ( MFI ) for each sample are shown in the table .

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

(G) FACS analysis of Gag positive cells from donor 2 in different conditions. Red: uninfected cells. Blue: untreated infected cells at 7 d p.i. as in B. Orange: cells were treated with NVP for 7 d as in C. Green: infected cells were exposed to NVP for 3 d, then NVP was washed out and cells were cultured for another 4 d as in D. Median of fluorescence intensities (MFI) for each sample are shown in the table.


a ) COS - 7 cells expressing EGFP - tagged wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin exon 1 ( green ) for 48 h stained for endogenous mTOR ( red ) show mTOR in mutant aggregates .

O O O O O O O O O O O O O O O O O O O O O O O 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) COS-7 cells expressing EGFP-tagged wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin exon 1 (green) for 48 h stained for endogenous mTOR (red) show mTOR in mutant aggregates.


( b ) Double - labeling immunohistochemistry of huntingtin ( EM48 ; green ) and mTOR ( red ) in brain tissue of mice expressing mutant huntingtin .

O O O O 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) Double-labeling immunohistochemistry of huntingtin (EM48; green) and mTOR (red) in brain tissue of mice expressing mutant huntingtin.


( c ) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease or unaffected controls using antibody to mTOR .

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

(c) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease or unaffected controls using antibody to mTOR.


( d ) Western blot of lysates from stable inducible PC12 cells 48 h after expression of wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin showing mTOR in the stacking gel ( mTOR insoluble ) in cells expressing mutant huntingtin .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot of lysates from stable inducible PC12 cells 48 h after expression of wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin showing mTOR in the stacking gel (mTOR insoluble) in cells expressing mutant huntingtin.


( e ) High - molecular - mass aggregates of mTOR in both PC12 ( i ) and COS - 7 ( ii ) cells expressing mutant ( Gln74 ; Q74 ) huntingtin . Blots of cell lysates from PC12 cells were probed with antibodies to phosphorylated mTOR ( Ser2448 ; p - mTOR ; iii ) and to GFP ( iv ) ; both showed insoluble material in the stack .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) High-molecular-mass aggregates of mTOR in both PC12 (i) and COS-7 (ii) cells expressing mutant (Gln74; Q74) huntingtin. Blots of cell lysates from PC12 cells were probed with antibodies to phosphorylated mTOR (Ser2448; p-mTOR; iii) and to GFP (iv); both showed insoluble material in the stack.


( f ) Cell lysates from stable inducible PC12 cells and transiently transfected COS - 7 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin for 48 h were immunoprecipitated using antibody to GFP and immunoblotted with antibody to mTOR . Uninduced PC12 cells with mutant huntingtin ( UI ) and untransfected COS - 7 cells ( UT ) were controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell lysates from stable inducible PC12 cells and transiently transfected COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin for 48 h were immunoprecipitated using antibody to GFP and immunoblotted with antibody to mTOR. Uninduced PC12 cells with mutant huntingtin (UI) and untransfected COS-7 cells (UT) were controls.


( g ) Brain lysates from mice expressing mutant huntingtin ( Tg ) or control ( Cont ) littermates were immunoprecipitated with antibody to mTOR and immunodetected with an antibody recognizing the polyglutamine tract ( IC2 ) . Htt ,

O O O O O O O O O O O O O O O O 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) Brain lysates from mice expressing mutant huntingtin (Tg) or control (Cont) littermates were immunoprecipitated with antibody to mTOR and immunodetected with an antibody recognizing the polyglutamine tract (IC2). Htt, huntingtin


( a ) Reduced levels of soluble mTOR ( as a function of tubulin ) in stable inducible PC12 cells expressing mutant ( Gln74 ; Q74 ) huntingtin compared with those expressing wild - type ( Gln23 ; Q23 ) huntingtin ( four independent experiments ; P 0 . 0001 ; unpaired t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Reduced levels of soluble mTOR (as a function of tubulin) in stable inducible PC12 cells expressing mutant (Gln74; Q74) huntingtin compared with those expressing wild-type (Gln23; Q23) huntingtin (four independent experiments; P 0.0001; unpaired t-test).


( b ) Reduced levels of soluble mTOR in brain lysates from mice expressing mutant huntingtin ( M1 , M2 ; n = 9 ) compared with age - matched wild - type littermates ( C1 , C2 ; n = 9 ; P 0 . 001 , unpaired t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Reduced levels of soluble mTOR in brain lysates from mice expressing mutant huntingtin (M1, M2; n = 9) compared with age-matched wild-type littermates (C1, C2; n = 9; P 0.001, unpaired t-test).


( c ) COS - 7 cells transiently transfected with wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin ( green ) labeled for endogenous 4E - BP1 ( red ) .

O O O O O O O O O O O O O O O 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) COS-7 cells transiently transfected with wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) labeled for endogenous 4E-BP1 (red).


( d ) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease showed 4E - BP1 in intranuclear inclusions . No 4E - BP1 - stained inclusions were seen in controls ( data not shown ) . (

(d) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease showed 4E-BP1 in intranuclear inclusions. No 4E-BP1-stained inclusions were seen in controls (data not shown). (


( e ) Western blot of cell lysates from stable inducible PC12 cells transfected with wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin shows that cells expressing mutant huntingtin had 31 % ( ± 4 . 3 % s . e . ) less phosphorylated S6K1 ( top ) relative to total S6K1 ( bottom ) than cells expressing wild - type huntingtin . ( f ) Similarly , cells expressing mutant ( Gln74 ; Q74 ) huntingtin had 30 % ( ± 4 . 5 % s . d . ) less phosphorylated 4E - BP1 ( top ) relative to total 4E - BP1 ( bottom ) than cells expressing wild - type ( Gln23 ; Q23 ) huntingtin . Expression of wild - type and mutant huntingtin was induced for 48 h ( e , f ) . ( g ) Cells expressing mutant ( Gln74 ; Q74 ) huntingtin also had lower levels of phosphorylated forms of transiently transfected FLAG - tagged 4E - BP1 than cells expressing wild - type ( Gln23 ; Q23 ) huntingtin . The multiple bands in 4E - BP1 ( f , g ) are due to its different phosphorylated species .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot of cell lysates from stable inducible PC12 cells transfected with wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin shows that cells expressing mutant huntingtin had 31% (± 4.3% s.e.) less phosphorylated S6K1 (top) relative to total S6K1 (bottom) than cells expressing wild-type huntingtin. (f) Similarly, cells expressing mutant (Gln74; Q74) huntingtin had 30% (± 4.5% s.d.) less phosphorylated 4E-BP1 (top) relative to total 4E-BP1 (bottom) than cells expressing wild-type (Gln23; Q23) huntingtin. Expression of wild-type and mutant huntingtin was induced for 48 h (e,f). (g) Cells expressing mutant (Gln74; Q74) huntingtin also had lower levels of phosphorylated forms of transiently transfected FLAG-tagged 4E-BP1 than cells expressing wild-type (Gln23; Q23) huntingtin. The multiple bands in 4E-BP1 (f,g) are due to its different phosphorylated species.


( a ) COS - 7 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin ( green ) were labeled for S6 phosphorylated at Ser235 and Ser236 ( red ) . Percentages of cells expressing wild - type or mutant huntingtin that did ( + agg ) or did not ( − agg ) contain aggregates were scored for negative staining for phosphorylated S6 ( p - S6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) were labeled for S6 phosphorylated at Ser235 and Ser236 (red). Percentages of cells expressing wild-type or mutant huntingtin that did (+agg) or did not (−agg) contain aggregates were scored for negative staining for phosphorylated S6 (p-S6).


( b ) Brain sections from control ( WT ) and mice expressing mutant huntingtin ( HD ) were stained for phosphorylated S6 ( red ) and with antibody to huntingtin ( EM48 ; green ) .

O O O O O O O O O O O O O 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) Brain sections from control (WT) and mice expressing mutant huntingtin (HD) were stained for phosphorylated S6 (red) and with antibody to huntingtin (EM48; green).


( c , d ) Impaired mTOR - dependent TOP translation in cells expressing mutant huntingtin . ( c ) PC12 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin were transfected with luciferase constructs containing 5 ′ untranslated sequence with ( + ) or without ( − ) a TOP sequence along with β - gal . Each bar represents the difference between the luciferase activities in the presence versus the absence of rapamycin . Data shown are from one representative experiment ( of three ) done in sextuplicate ( error bars represent s . e . ; raw data are given in Supplementary Fig . 4 online ) . ( d ) COS - 7 cells were cotransfected with wild - type or mutant huntingtin , a nontypical TOP or control luciferase vector and β - gal . The experiment was done and analyzed as in c ( raw data are given in Supplementary Fig . 4 online ) ( e - g ) Relevance of mTOR sequestration to other polyglutamine expansion

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

(c,d) Impaired mTOR-dependent TOP translation in cells expressing mutant huntingtin. (c) PC12 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin were transfected with luciferase constructs containing 5′ untranslated sequence with (+) or without (−) a TOP sequence along with β-gal. Each bar represents the difference between the luciferase activities in the presence versus the absence of rapamycin. Data shown are from one representative experiment (of three) done in sextuplicate (error bars represent s.e.; raw data are given in Supplementary Fig. 4 online). (d) COS-7 cells were cotransfected with wild-type or mutant huntingtin, a nontypical TOP or control luciferase vector and β-gal. The experiment was done and analyzed as in c (raw data are given in Supplementary Fig. 4 online) (e-g) Relevance of mTOR sequestration to other polyglutamine expansion diseases


( e ) COS - 7 cells expressing Xpress - tagged ataxin - 1 with 92 polyglutamine repeats ( Q92 ) for 48 h were doubly labeled with antibodies to Xpress ( green ) and to mTOR ( red ) .

O O O O O O O O O O O O O O O O O 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) COS-7 cells expressing Xpress-tagged ataxin-1 with 92 polyglutamine repeats (Q92) for 48 h were doubly labeled with antibodies to Xpress (green) and to mTOR (red).


( f ) mTOR immunohistochemistry in brains of individuals with dentatorubral - pallidoluysian atrophy ( DRPLA ) and spinocerebellar ataxia types 2 , 3 and 7 ( SCA2 , SCA3 and SCA7 ) .

O O O O O O O O O O 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) mTOR immunohistochemistry in brains of individuals with dentatorubral-pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia types 2, 3 and 7 (SCA2, SCA3 and SCA7).


( g ) Staining for phosphorylated S6 ( red ) in COS - 7 cells transiently transfected with mutant ( ataxin Q92 ; green ) or wild - type ( ataxin Q2 ) ataxin - 1 constructs . Results were significant and similar to those in a . Arrows in a , b , e and g show cells containing aggregates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Staining for phosphorylated S6 (red) in COS-7 cells transiently transfected with mutant (ataxin Q92; green) or wild-type (ataxin Q2) ataxin-1 constructs. Results were significant and similar to those in a. Arrows in a, b, e and g show cells containing aggregates.


( a ) LC3 - II levels , which closely correlate with autophagic activity , were higher in mutant lines ( expressing Gln74 huntingtin ; Q74 ; lanes 3 and 4 ) than in wild - type lines ( expressing Gln23 huntingtin ; Q23 ; lanes 1 and 2 ) . Actin was used as a loading control . Data are shown for two independent clonal lines expressing mutant or wild - type huntingtin . Quantification of the band intensity is shown in the 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 O O O O O O O O O O O O

(a) LC3-II levels, which closely correlate with autophagic activity, were higher in mutant lines (expressing Gln74 huntingtin; Q74; lanes 3 and 4) than in wild-type lines (expressing Gln23 huntingtin; Q23; lanes 1 and 2). Actin was used as a loading control. Data are shown for two independent clonal lines expressing mutant or wild-type huntingtin. Quantification of the band intensity is shown in the right panel.


( b ) Immunofluorescence analysis of COS - 7 cells expressing wild - type ( Gln23 ; Q23 ) or mutant ( Gln74 ; Q74 ) huntingtin with antibody to LC3 showed more autophagic vacuoles in cells expressing mutant huntingtin that had aggregates ( arrow ) . COS - 7 cells without the primary antibody ( negative ) and COS - 7 cells without any treatment ( untreated ) stained for LC3 are also shown . The numbers of EGFP - positive cells expressing wild - type or mutant huntingtin with ( + agg ) and without ( − agg ) aggregates that had higher numbers of autophagic vacuoles ( > 15 - 20 vesicles per cell ) is shown in the graph . COS - 7 cells grown in starvation medium ( starvation ) or treated with rapamycin ( Rap ) for 4 h to induce autophagy had more autophagic vacuoles ( arrows ) .

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

(b) Immunofluorescence analysis of COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin with antibody to LC3 showed more autophagic vacuoles in cells expressing mutant huntingtin that had aggregates (arrow). COS-7 cells without the primary antibody (negative) and COS-7 cells without any treatment (untreated) stained for LC3 are also shown. The numbers of EGFP-positive cells expressing wild-type or mutant huntingtin with (+agg) and without (−agg) aggregates that had higher numbers of autophagic vacuoles (>15-20 vesicles per cell) is shown in the graph. COS-7 cells grown in starvation medium (starvation) or treated with rapamycin (Rap) for 4 h to induce autophagy had more autophagic vacuoles (arrows).


( a ) Activation of mTOR activity with rheb increases aggregation and toxicity of mutant ( Gln74 ; Q74 ) huntingtin . COS - 7 cells were cotransfected with a mammalian expression vector encoding rheb ( or empty vector control ) and mutant huntingtin for 48 h at a 3 : 1 mass ratio ( rheb or empty vector to huntingtin ) to ensure that almost all cells expressing huntingtin also expressed rheb . The percentages of EGFP - positive cells with aggregates or apoptotic nuclear morphology ( as described previously ) were markedly higher when rheb was overexpressed . Control cells express mutant huntingtin ( green ) without rheb . The middle panels show images captured at the same comparatively low gain to distinguish aggregates from soluble EGFP . The left panels show the same fields at a similar higher gain to indicate the total number of EGFP - positive cells ( this shows that the effect seen in the middle panels is not due to loss of cells ) . Nuclear staining with DAPI ( blue ) is shown in the right panels . * * * P 0 . 0001 . Error bars represent s . e . for a representative triplicate experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Activation of mTOR activity with rheb increases aggregation and toxicity of mutant (Gln74; Q74) huntingtin. COS-7 cells were cotransfected with a mammalian expression vector encoding rheb (or empty vector control) and mutant huntingtin for 48 h at a 3:1 mass ratio (rheb or empty vector to huntingtin) to ensure that almost all cells expressing huntingtin also expressed rheb. The percentages of EGFP-positive cells with aggregates or apoptotic nuclear morphology (as described previously) were markedly higher when rheb was overexpressed. Control cells express mutant huntingtin (green) without rheb. The middle panels show images captured at the same comparatively low gain to distinguish aggregates from soluble EGFP. The left panels show the same fields at a similar higher gain to indicate the total number of EGFP-positive cells (this shows that the effect seen in the middle panels is not due to loss of cells). Nuclear staining with DAPI (blue) is shown in the right panels. ***P 0.0001. Error bars represent s.e. for a representative triplicate experiment.


( b , c ) Treatment with rapamycin rescues huntingtin - induced degeneration in flies 2 d after eclosion . Photographs of ommatidia of flies expressing mutant ( Gln120 ; Q120 ) huntingtin treated with DMSO or rapamycin from the larval stage into adulthood . More rhabdomeres are visible in the ommatidia of flies treated with rapamycin . This effect is significant ( P 0 . 0001 ; Mann - Whitney U test ) . At 2 d after eclosion , there is no reduction in the number of visible rhabdomeres in wild - type untreated flies ( data not shown ) .

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 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

(b,c) Treatment with rapamycin rescues huntingtin-induced degeneration in flies 2 d after eclosion. Photographs of ommatidia of flies expressing mutant (Gln120; Q120) huntingtin treated with DMSO or rapamycin from the larval stage into adulthood. More rhabdomeres are visible in the ommatidia of flies treated with rapamycin. This effect is significant (P 0.0001; Mann-Whitney U test). At 2 d after eclosion, there is no reduction in the number of visible rhabdomeres in wild-type untreated flies (data not shown).


Open bars , mice treated with a placebo ; filled bars , mice treated with CCI - 779 . ( a - c ) Behavioral tests in mice expressing mutant huntingtin after treatment at 16 weeks ( CCI - 779 , n = 14 ; placebo , n = 14 ) , 18 weeks ( CCI - 779 , n = 10 ; placebo , n = 9 ) , 20 weeks ( CCI - 779 , n = 9 ; placebo , n = 7 ) and 22 weeks ( CCI - 779 , n = 5 ; placebo , n = 6 ) of age . Scores are explained in Methods . Data for all time points are given in Supplementary Figure 13 online . ( a ) Tremors . Overall effect from all treated time points : P = 0 . 0006 , odds ratio ( OR ) = 0 . 21 , 95 % confidence interval ( 95 % c . i . ) = 0 . 098 - 0 . 45 . 16 weeks , P = 0 . 01 ; 18 weeks , P = 0 . 08 ; 20 weeks , P = 0 . 14 ; 22 weeks , P = 0 . 07 . ( b ) Wire maneuver . Overall effect from all treated time points : P = 0 . 02 , OR = 0 . 51 , 95 % c . i . = 0 . 29 - 0 . 92 . 16 weeks , P = 0 . 29 ; 18 weeks , P = 0 . 40 ; 20 weeks , P = 0 . 003 ; 22 weeks , P = 0 . 008 . ( c ) Grip strength . Overall effect from all treated time points : P 0 . 0001 OR = 12 . 73 , 95 % c . i . = 4 . 35 - 37 . 2 . 16 weeks , P = 0 . 1827 ; 18 weeks , P = 0 . 0412 ; 20 weeks , P = 0 . 0021 ; 22 weeks , P = 0 . 0285 .

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

Open bars, mice treated with a placebo; filled bars, mice treated with CCI-779. (a-c) Behavioral tests in mice expressing mutant huntingtin after treatment at 16 weeks (CCI-779, n = 14; placebo, n = 14), 18 weeks (CCI-779, n = 10; placebo, n = 9), 20 weeks (CCI-779, n = 9; placebo, n = 7) and 22 weeks (CCI-779, n = 5; placebo, n = 6) of age. Scores are explained in Methods. Data for all time points are given in Supplementary Figure 13 online. (a) Tremors. Overall effect from all treated time points: P = 0.0006, odds ratio (OR) = 0.21, 95% confidence interval (95% c.i.) = 0.098-0.45. 16 weeks, P = 0.01; 18 weeks, P = 0.08; 20 weeks, P = 0.14; 22 weeks, P = 0.07. (b) Wire maneuver. Overall effect from all treated time points: P = 0.02, OR = 0.51, 95% c.i. = 0.29-0.92. 16 weeks, P = 0.29; 18 weeks, P = 0.40; 20 weeks, P = 0.003; 22 weeks, P = 0.008. (c) Grip strength. Overall effect from all treated time points: P 0.0001 OR = 12.73, 95% c.i. = 4.35-37.2. 16 weeks, P = 0.1827; 18 weeks, P = 0.0412; 20 weeks, P = 0.0021; 22 weeks, P = 0.0285.


( d ) Accelerating rotarod tests in mice expressing mutant huntingtin after treatment at 4 weeks , ( CCI - 779 , n = 16 ; placebo , n = 17 ) , 14 weeks ( CCI - 779 , n = 11 ; placebo , n = 10 ) , 16 weeks ( CCI - 779 , n = 14 ; placebo , n = 14 ) and 18 weeks ( CCI - 779 , n = 10 ; placebo , n = 9 ) of age . Overall effect from all treated time points : P = 0 . 035 . 4 weeks , P = 0 . 227 ; 14 weeks , P = 0 . 012 ; 16 weeks , P = 0 . 0025 ; 18 weeks , P = 0 . 057 . CCI - 779 had no discernable effects on the performance of nontransgenic mice on any of these behavioral tests ( data not shown ) . There was no difference in the male : female ratios of the groups treated with placebo or with CCI - 779 .

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

(d) Accelerating rotarod tests in mice expressing mutant huntingtin after treatment at 4 weeks, (CCI-779, n = 16; placebo, n = 17), 14 weeks (CCI-779, n = 11; placebo, n = 10), 16 weeks (CCI-779, n = 14; placebo, n = 14) and 18 weeks (CCI-779, n = 10; placebo, n = 9) of age. Overall effect from all treated time points: P = 0.035. 4 weeks, P = 0.227; 14 weeks, P = 0.012; 16 weeks, P = 0.0025; 18 weeks, P = 0.057. CCI-779 had no discernable effects on the performance of nontransgenic mice on any of these behavioral tests (data not shown). There was no difference in the male:female ratios of the groups treated with placebo or with CCI-779.


( a ) Weights of mice expressing mutant huntingtin ( Tg ) and nontransgenic ( NT ) littermates treated with CCI - 779 or with a placebo . ( b ) Brain weights of mice expressing mutant huntingtin treated with CCI - 779 ( black bars ) or with a placebo ( white bars ) and nontransgenic untreated littermates ( gray bars ) at 8 weeks ( CCI - 779 , n = 3 ; placebo , n = 3 ; nontransgenic , n = 7 ; CCI - 779 versus placebo , P = 0 . 023 ) , 12 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 5 ; CCI - 779 versus placebo , P = 0 . 90 ) and 16 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 2 ; CCI - 779 versus placebo , P = 0 . 22 ) of age . ( c ) Ratio of brain weight to body weight of mice expressing mutant huntingtin treated with CCI - 779 ( black bars ) or with a placebo ( white bars ) and nontransgenic untreated littermates ( gray bars ) at 8 weeks ( CCI - 779 , n = 3 ; placebo , n = 3 ; nontransgenic , n = 7 ; CCI - 779 versus placebo , P = 0 . 06 ) , 12 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 5 ; CCI - 779 versus placebo , P = 0 . 38 ) and 16 weeks ( CCI - 779 , n = 2 ; placebo , n = 3 ; nontransgenic , n = 2 ; CCI - 779 versus placebo , P = 0 . 56 ) of age .

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

(a) Weights of mice expressing mutant huntingtin (Tg) and nontransgenic (NT) littermates treated with CCI-779 or with a placebo. (b) Brain weights of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.023), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.90) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.22) of age. (c) Ratio of brain weight to body weight of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.06), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.38) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.56) of age.


( a ) CCI - 779 reduces aggregate load in mice expressing mutant huntingtin . Peroxidase immunohistochemistry of brains of mice expressing mutant huntingtin at 16 weeks of age . Mice treated with CCI - 779 had fewer aggregates per unit area in the striatum than mice treated with a placebo . Overall cell staining in the tissues of mice treated with CCI - 779 was weaker than in those treated with a placebo and in nontransgenic untreated littermates ( data not shown ) . Aggregates , when visible , were smaller in mice treated with CCI - 779 than in those treated with a placebo , as apparent in the higher magnification images of single cells with aggregate ( bottom panels ) . Six coronal sections per mouse were analyzed ; these corresponded to the bregma ± 1 mm . We counted aggregates in five different high - power fields ( 60 × objective ) per slide on each side of the brain , sampling fields from the dorsal to the ventral part of the striatum just underneath the external capsule . The striata of mice treated with CCI - 779 had 70 % fewer aggregates than the striata of mice treated with a placebo ( P = 0 . 0001 , two - tailed t - test ) .

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

(a) CCI-779 reduces aggregate load in mice expressing mutant huntingtin. Peroxidase immunohistochemistry of brains of mice expressing mutant huntingtin at 16 weeks of age. Mice treated with CCI-779 had fewer aggregates per unit area in the striatum than mice treated with a placebo. Overall cell staining in the tissues of mice treated with CCI-779 was weaker than in those treated with a placebo and in nontransgenic untreated littermates (data not shown). Aggregates, when visible, were smaller in mice treated with CCI-779 than in those treated with a placebo, as apparent in the higher magnification images of single cells with aggregate (bottom panels). Six coronal sections per mouse were analyzed; these corresponded to the bregma ± 1 mm. We counted aggregates in five different high-power fields (60× objective) per slide on each side of the brain, sampling fields from the dorsal to the ventral part of the striatum just underneath the external capsule. The striata of mice treated with CCI-779 had 70% fewer aggregates than the striata of mice treated with a placebo (P = 0.0001, two-tailed t-test).


( b ) CCI - 779 blocks mTOR signaling in vivo . Immunohistochemistry of mice with Huntington disease at 16 weeks of age with antibody to phosphorylated S6 , in the cortical area and striatum . Levels of phosphorylated S6 were lower in mice treated with CCI - 779 than in those treated with a placebo .

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(b) CCI-779 blocks mTOR signaling in vivo. Immunohistochemistry of mice with Huntington disease at 16 weeks of age with antibody to phosphorylated S6, in the cortical area and striatum. Levels of phosphorylated S6 were lower in mice treated with CCI-779 than in those treated with a placebo.


( D ) Electron microscopy images from purified 40S and 60S ribosomal complexes ( 1 pmol each ) assembled in vitro in the presence of MgCl2 , and in the presence or absence of 5 pmol of ( PR ) 20 . Assembled 80S particles are indicated ( red arrows ) . Scale bar ( white ) represents 10 nm . ( E ) Quantification of 80S particles identified in ( D ) ( n = 1000 ) in non - assembly ( 1 mM MgCl2 ) or assembly ( 5 mM MgCl2 ) conditions . (

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(D) Electron microscopy images from purified 40S and 60S ribosomal complexes (1 pmol each) assembled in vitro in the presence of MgCl2, and in the presence or absence of 5 pmol of (PR)20. Assembled 80S particles are indicated (red arrows). Scale bar (white) represents 10 nm. (E) Quantification of 80S particles identified in (D) (n=1000) in non-assembly (1 mM MgCl2) or assembly (5 mM MgCl2) conditions. (


( C , D ) High - throughput microscopy ( HTM ) - mediated analysis of 5 - ethynyl - uridine ( EU ) ( C ) and polyA + mRNA ( D ) levels per nucleus found in U2OS cells exposed to 10 μM ( PR ) 20 for 16 h . EU was added 30 ´ prior to fixation ( n = 3 ) . Black lines indicate median values in both panels . In D , boxes define upper and lower quartiles and whiskers mark the highest and lowest observations . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - test . Representative images from these analyses are provided in each case . Scale bar ( white ) indicates 2 - 5 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 B-MEASURED_VAR 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 I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) High-throughput microscopy (HTM)-mediated analysis of 5-ethynyl-uridine (EU) (C) and polyA+ mRNA (D) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 16 h. EU was added 30´ prior to fixation (n=3). Black lines indicate median values in both panels. In D, boxes define upper and lower quartiles and whiskers mark the highest and lowest observations. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.


( E ) Effect of ( PR ) 20 ( 10 μM , 16 h ) in the number of Cajal Bodies ( CB ) identified by immunofluorescence with anti - COILIN and anti - SMN antibodies ( n = 3 ) . Data represent mean values ± SD . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - test . Representative images from these analyses are provided in each case . Scale bar ( white ) indicates 2 - 5 μm .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of (PR)20 (10 μM, 16 h) in the number of Cajal Bodies (CB) identified by immunofluorescence with anti-COILIN and anti-SMN antibodies (n=3). Data represent mean values ± SD. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.


( B , C ) Percentage of GAPDH or 5 . 8 rRNA levels quantified by qPCR in reactions containing increasing doses of ( PR ) 20 ( n = 3 ) . Data represent mean values ± SEM . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - test .

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(B, C) Percentage of GAPDH or 5.8 rRNA levels quantified by qPCR in reactions containing increasing doses of (PR)20 (n=3).Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test.


( D ) HTM - mediated analysis of 5 - ethynyl - deoxyuridine ( EdU ) levels per nucleus found in U2OS cells exposed to 10 μM ( PR ) 20 for 4 h . EdU was added 30 ´ prior to fixation . Representative images from these analyses are provided ( right ) . Black lines indicate median values . Scale bar ( white ) indicates 5 μm .

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(D) HTM-mediated analysis of 5-ethynyl-deoxyuridine (EdU) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 4 h. EdU was added 30´ prior to fixation. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.


( E ) HTM - mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM ( PR ) 20 for the indicated times . Note the accumulation of cells with persistent DNA damage ( γH2AX ) 10 h after IR in the presence of the DPR . Representative images from these analyses are provided ( right ) . Black lines indicate median values . Scale bar ( white ) indicates 5 μm .

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(E) HTM-mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM (PR)20 for the indicated times. Note the accumulation of cells with persistent DNA damage (γH2AX) 10 h after IR in the presence of the DPR. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.


( D ) Immunofluorescence of Cy3 - labelled salmon protamine ( red ) and the nucleolar factor UBF1 ( green ) in U2OS cells treated with 30 μM Cy3 - protamine for 4 h . Scale bar ( white ) indicates 2 . 5 μm .

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(D) Immunofluorescence of Cy3-labelled salmon protamine (red) and the nucleolar factor UBF1 (green) in U2OS cells treated with 30 μM Cy3-protamine for 4 h. Scale bar (white) indicates 2.5 μm.


( E , F ) HTM - mediated quantification of the EU levels per nucleus ( E ) and O - propargyl - puromycin ( OPP ) levels per cell ( F ) in U2OS cells exposed to 30 μM protamine for 16 ( E ) or 24 ( F ) h . EU and OPP were added 30 ´ prior to fixation . Black lines indicate median values ( * * * * , p < 0 . 0001 ; t - test ) .

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(E, F) HTM-mediated quantification of the EU levels per nucleus (E) and O-propargyl-puromycin (OPP) levels per cell (F) in U2OS cells exposed to 30 μM protamine for 16 (E) or 24 (F) h. EU and OPP were added 30´ prior to fixation. Black lines indicate median values (****, p<0.0001; t-test).


( D ) Immunofluorescence of HA - ( PR ) 20 ( green ) and the nucleolar factor UBF1 ( red ) in U2OS cells treated with 7 . 5 μM HA - ( PR ) 20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h . Data information : Scale bar ( white ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - test .

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(D) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( E ) Immunofluorescence of HA - ( PR ) 20 ( green ) and Cy5 ( red ) in U2OS cells treated with 7 . 5 μM HA - ( PR ) 20 alone or together with 4 μM of Cy5 - labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h . Data information : Scale bar ( white ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - test .

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(E) Immunofluorescence of HA-(PR)20 (green) and Cy5 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 4 μM of Cy5-labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( F ) Clonogenic survival assay of U2OS cells exposed to 7 . 5 μM with the indicated peptides or protamine ( PROT . ) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0 . 5 μM Heparin .

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(F) Clonogenic survival assay of U2OS cells exposed to 7.5 μM with the indicated peptides or protamine (PROT.) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0.5 μM Heparin.


( G ) Immunofluorescence of HA - ( PR ) 20 ( green ) and the nucleolar factor UBF1 ( red ) in U2OS cells treated with 7 . 5 μM HA - ( PR ) 20 alone or together with heparin ( 0 . 5 μM ) . Data information : Scale bar ( white ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - test .

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(G) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with heparin (0.5 μM). Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( A ) Immunofluorescence of the neuronal marker TUBB3 ( red ) in mouse motor neurons differentiated from mESC ( mMN ) or from NSC34 cells ( NSC34DIFF ) exposed to 2 . 5 μM ( PR ) 20 for 24 and 48 hours , respectively , with or without 2 μM of a non - coding 38nt oligonucleotide . Nuclei were stained with DAPI ( blue ) . Scale bar ( white ) , 10 μm . Note that besides its impact on overall survival , the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide . ( B , C ) Cell viability quantified CellTiter - Glo luminescence assays mMN ( B ) and NSC34DIFF ( C ) treated as in ( A ) ( n = 3 ) . Data represent mean values ± SEM . ( Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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(A) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without 2 μM of a non-coding 38nt oligonucleotide. Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. Note that besides its impact on overall survival, the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide. (B,C) Cell viability quantified CellTiter-Glo luminescence assays mMN (B) and NSC34DIFF (C) treated as in (A) (n=3). Data represent mean values ± SEM. ( Data information: **, p<0.01; ***, p<0.001.


( D ) Immunofluorescence of the neuronal marker TUBB3 ( red ) in mouse motor neurons differentiated from mESC ( mMN ) or from NSC34 cells ( NSC34DIFF ) exposed to 2 . 5 μM ( PR ) 20 for 24 and 48 hours , respectively , with or without heparin ( 0 . 5 μM ) . Nuclei were stained with DAPI ( blue ) . Scale bar ( white ) , 10 μm . ( E , F ) Cell viability quantified CellTiter - Glo luminescence assays mMN ( E ) and NSC34DIFF ( F ) treated as in ( D ) ( n = 3 ) . Data represent mean values ± SEM . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 . S

O O O O O O O O O O O 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 I-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without heparin (0.5 μM). Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. (E, F) Cell viability quantified CellTiter-Glo luminescence assays mMN (E) and NSC34DIFF (F) treated as in (D) (n=3). Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001. S


( B ) USH1C transcripts were consistently detected in all tested organs of WT and USH1C pigs , except in the heart and cerebellum . RT - PCR for GAPDH was used as control ( Ctrl . ) .

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(B) USH1C transcripts were consistently detected in all tested organs of WT and USH1C pigs, except in the heart and cerebellum. RT-PCR for GAPDH was used as control (Ctrl.).


( D ) Representative Western blot analysis proved lack of harmonin protein expression in retina and duodenum of USH1C pigs at the age of 3 weeks ( 3w ) ( 1 piglet , 1 retina , 1 duodenum ) and 1 year ( 1y ) ( 2 pigs , 1 retina each , 3 TRs ) . Arrow indicates the expected size of harmonin isoform a ( 72kDa ) .

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(D) Representative Western blot analysis proved lack of harmonin protein expression in retina and duodenum of USH1C pigs at the age of 3 weeks (3w) (1 piglet, 1 retina, 1 duodenum) and 1 year (1y) (2 pigs, 1 retina each, 3 TRs). Arrow indicates the expected size of harmonin isoform a (72kDa).


( E ) Representative immunofluorescence staining of harmonin ( green ) of longitudinal cryosection through WT and USH1C pig retinas ( 2 pigs , 1y , 1 retina each , 3 TRs , scale bar 10µm ) demonstrated the absence of harmonin staining in the outer segment ( OS ) of photoreceptor cells and in the other layers below the outer limiting membrane ( OLM , arrow ) of the USH1C pig retina .

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(E) Representative immunofluorescence staining of harmonin (green) of longitudinal cryosection through WT and USH1C pig retinas (2 pigs, 1y, 1 retina each, 3 TRs, scale bar 10µm) demonstrated the absence of harmonin staining in the outer segment (OS) of photoreceptor cells and in the other layers below the outer limiting membrane (OLM, arrow) of the USH1C pig retina.


( F ) Representative Western blot analysis of retinal protein extracts , indicating that the abundance of the USH proteins SANS ( USH1G ) , whirlin ( USH2D ) or myosin7a ( USH1B ) is unaffected ( 1 piglet , 3w , 1 retina each , 2 TRs ) .

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(F) Representative Western blot analysis of retinal protein extracts, indicating that the abundance of the USH proteins SANS (USH1G), whirlin (USH2D) or myosin7a (USH1B) is unaffected (1 piglet, 3w, 1 retina each, 2 TRs).


( A , B ) Upregulation of GFAP in the retina of USH1C pigs at an age of 3 weeks ( 3w ) . ( A ) Representative immunofluorescence staining of GFAP in Müller glia cells which extend throughout almost the entire retina from the OLM ( arrow ) to the ganglion cell layer ( GCL ) of the retina . The consistent increase of GFAP expression in the Müller glia of USH1C pigs indicates Müller cell activation and gliosis . IS , inner segment ; ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; scale bar : 20 µm . ( B ) Left panel : Western blot analysis of GFAP protein expression in 3w old USH1C piglets and age matched controls . Anti - actin Western blot was used as loading control . Right panel : Quantification of Western blot bands in 4 gels by the LI - COR Odyssey system revealed a strong increase GFAP expression in 3w USH1C piglets when compared to age - matched WT controls ( 2 piglets , 3w , 2 retinas each , 2 TRs , error bars represent standard deviation ( SD ) of the mean , two - tailed Student ' s t tests , * p < 0 . 05 ) .

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(A, B) Upregulation of GFAP in the retina of USH1C pigs at an age of 3 weeks (3w). (A) Representative immunofluorescence staining of GFAP in Müller glia cells which extend throughout almost the entire retina from the OLM (arrow) to the ganglion cell layer (GCL) of the retina. The consistent increase of GFAP expression in the Müller glia of USH1C pigs indicates Müller cell activation and gliosis. IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; scale bar: 20 µm. (B) Left panel: Western blot analysis of GFAP protein expression in 3w old USH1C piglets and age matched controls. Anti-actin Western blot was used as loading control. Right panel: Quantification of Western blot bands in 4 gels by the LI-COR Odyssey system revealed a strong increase GFAP expression in 3w USH1C piglets when compared to age-matched WT controls (2 piglets, 3w, 2 retinas each, 2 TRs, error bars represent standard deviation (SD) of the mean, two-tailed Student's t tests, *p<0.05).


( C , D ) Reduced synaptic width in USH1C pigs at an age of 1 year ( 1y ) . ( C ) Fluorescent microscopic analysis of the cone synapse phenotype . Left panel : Representative images of longitudinal sections through WT and USH1C pig retinae stained for the pre - synaptic marker PSD - 95 ( green ) and by fluorescent peanut agglutinin ( PNA , red ) for cone synaptic pedicles ( white arrows ) and counter - stained by DAPI for nuclear DNA ( blue ) . Scale bar 10µm . Middle panel : Higher magnification of a PNA - stained cone synaptic pedicle . Synapse width was determined as the maximum extension of consistent PNA signals . Scale bar 1µm . Right panel : Measuring cone synaptic pedicle width in WT and USH1C pigs by applying a Fiji script to PNA - stained sections indicated reduced synaptic width ( 2 pigs , 1y , 1 retina each , number of examined synapses indicated , error bars represent SD , two - tailed Student ' s t tests , * * * p < 0 . 001 ) . ( D ) Determining cone synaptic pedicles width by TEM . Left panel : Representative images of retinal cross sections . Scale bar 500nm . Right panel : Quantification of synaptic width confirmed the significantly reduced width of cone synaptic pedicles in USH1C pigs . ( 1 pig , 1y , 1 retina each , number of examined synapses indicated ,

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

(C, D) Reduced synaptic width in USH1C pigs at an age of 1 year (1y). (C) Fluorescent microscopic analysis of the cone synapse phenotype. Left panel: Representative images of longitudinal sections through WT and USH1C pig retinae stained for the pre-synaptic marker PSD-95 (green) and by fluorescent peanut agglutinin (PNA, red) for cone synaptic pedicles (white arrows) and counter-stained by DAPI for nuclear DNA (blue). Scale bar 10µm. Middle panel: Higher magnification of a PNA-stained cone synaptic pedicle. Synapse width was determined as the maximum extension of consistent PNA signals. Scale bar 1µm. Right panel: Measuring cone synaptic pedicle width in WT and USH1C pigs by applying a Fiji script to PNA-stained sections indicated reduced synaptic width (2 pigs, 1y, 1 retina each, number of examined synapses indicated, error bars represent SD, two-tailed Student's t tests, ***p<0.001). (D) Determining cone synaptic pedicles width by TEM. Left panel: Representative images of retinal cross sections. Scale bar 500nm. Right panel: Quantification of synaptic width confirmed the significantly reduced width of cone synaptic pedicles in USH1C pigs. (1 pig, 1y, 1 retina each, number of examined synapses indicated,


( A - C ) Ciliary length measured by fluorescence microscopic analysis at 1 year of age ( 1y ) . ( A ) Representative immunostaining of the ciliary marker Gt335 ( green ) in longitudinal cryosections of PRC ciliary regions in WT and USH1C retinas . Counterstaining by DAPI , scale bar 10µm . ( B ) Gt335 staining at higher magnification and principle of quantifying ciliary length by Gt335 staining . Scale bar 500nm . ( C ) Length of connecting cilia ( CC ) appear elongated in USH1C when applying a Fiji script to immunostained histological sections ( 2 pigs , 1y , 1 retina each , number of examined connecting cilia is indicated , error bars represent SD , two - tailed Student ' s t tests , * * * * p < 0 . 0001 ) .

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

(A-C) Ciliary length measured by fluorescence microscopic analysis at 1 year of age (1y). (A) Representative immunostaining of the ciliary marker Gt335 (green) in longitudinal cryosections of PRC ciliary regions in WT and USH1C retinas. Counterstaining by DAPI, scale bar 10µm. (B) Gt335 staining at higher magnification and principle of quantifying ciliary length by Gt335 staining. Scale bar 500nm. (C) Length of connecting cilia (CC) appear elongated in USH1C when applying a Fiji script to immunostained histological sections (2 pigs, 1y, 1 retina each, number of examined connecting cilia is indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).


( D , E ) Measuring CC length in TEM sections . ( D ) Representative images of longitudinal cross sections and principle of determining CC length . Scale bar 500nm . ( E ) Elongation of CC length is confirmed in TEM sections of USH1C PRC . ( 1 pig , 1y , 1 retina each , number of examined connecting cilia indicated , error bars represent SD , two - tailed Student ' s t tests , * * * * p < 0 . 0001 ) .

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

(D, E) Measuring CC length in TEM sections. (D) Representative images of longitudinal cross sections and principle of determining CC length. Scale bar 500nm. (E) Elongation of CC length is confirmed in TEM sections of USH1C PRC. (1 pig, 1y, 1 retina each, number of examined connecting cilia indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).


Length of primary cilia in dermal fibroblasts . ( F ) Staining of ciliary structures in fibroblasts stained with ARL13B ( ciliary shaft , green ) and PCNT2 ( ciliary base , red ) , DAPI ( nucleus , blue ) . Scale bar upper panel 5 µm , lower panel 2 . 5 µm . ( H ) Similarly , ciliary length of fibroblasts from a human USH1CR31X / R80fs patient are elongated , compared to a healthy control ( 7 TR , number of cells examined indicated , KS - test , * * * * p < 0 . 0001 ) .

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

Length of primary cilia in dermal fibroblasts. (F) Staining of ciliary structures in fibroblasts stained with ARL13B (ciliary shaft, green) and PCNT2 (ciliary base, red), DAPI (nucleus, blue). Scale bar upper panel 5 µm, lower panel 2.5 µm. (H) Similarly, ciliary length of fibroblasts from a human USH1CR31X/R80fs patient are elongated, compared to a healthy control (7 TR, number of cells examined indicated, KS-test, ****p<0.0001).


( I - M ) Outer segment ( OS ) PRC architecture by TEM of longitudinal retinal sections . ( I ) At 3w , WT show normal sub - cellular organization , including parallel stacking of photosensitive discs and calyceal processes ( black arrows , representative image from 2 pigs , 2 retinas , scale bar 600nm ) . ( J ) In 3w USH1C rods , membrane discs appear also in vertical orientation ( white arrows , representative image from 2 pigs , 2 retinas , scale bar 750nm ) . At 1y , interstitial gaps appeared in disc stacks of the OS in USH1C pigs ( K , arrow , scale bar 550nm ) and vesicles are found at the OS base ( L , asterisk , scale bar 500nm ) . ( M ) The disc architecture in 1y animals was substantially distorted ( arrows , representative image from 2 pigs , 2 retinas , scale bar 850nm ) .

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

(I-M) Outer segment (OS) PRC architecture by TEM of longitudinal retinal sections. (I) At 3w, WT show normal sub-cellular organization, including parallel stacking of photosensitive discs and calyceal processes (black arrows, representative image from 2 pigs, 2 retinas, scale bar 600nm). (J) In 3w USH1C rods, membrane discs appear also in vertical orientation (white arrows, representative image from 2 pigs, 2 retinas, scale bar 750nm). At 1y, interstitial gaps appeared in disc stacks of the OS in USH1C pigs (K, arrow, scale bar 550nm) and vesicles are found at the OS base (L, asterisk, scale bar 500nm). (M) The disc architecture in 1y animals was substantially distorted (arrows, representative image from 2 pigs, 2 retinas, scale bar 850nm).


( A ) AAV transduction of retinal layers . AAV8 , AAV9 and Anc80 encoding eGFP under the ubiquitous CMV promoter were injected subretinally into the eyes of mature WT pigs in vivo . Upper Panel : Epifluorescence of dissected eyes demonstrates eGFP fluorescence in the bleb region of whole mount preparations of all three AAV samples . Dashed circles indicate bleb size . Lower panel : Epifluorescence images of longitudinal retina sections revealed eGFP expression in the PRC layer ( OS , IS and ONL ) for all AAV . Increased exposure ( 4x ) documented transduction of MGC ( arrows ) by AAV9 and Anc80 but not by AAV8 ( representative images from 1 pig , 1 retina each , 2 TR , 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 O O O O O O O O O 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) AAV transduction of retinal layers. AAV8, AAV9 and Anc80 encoding eGFP under the ubiquitous CMV promoter were injected subretinally into the eyes of mature WT pigs in vivo. Upper Panel: Epifluorescence of dissected eyes demonstrates eGFP fluorescence in the bleb region of whole mount preparations of all three AAV samples. Dashed circles indicate bleb size. Lower panel: Epifluorescence images of longitudinal retina sections revealed eGFP expression in the PRC layer (OS, IS and ONL) for all AAV. Increased exposure (4x) documented transduction of MGC (arrows) by AAV9 and Anc80 but not by AAV8 (representative images from 1 pig, 1 retina each, 2 TR, scale bars 25 µm).


( D ) Efficacy of AAV gene therapy was examined in vivo by sub - retinal injection of Anc80 virus capsid expressing harmonin _ a1 under a ubiquitous promoter into a USH1C pig ( see also Figure EV5 ) . Left panel : Sub - retinal injection reconstituted harmonin expression in the AAV - treated eye but not in the sham - treated eye ( 3 TR ) . Protein extract from a WT retina served as positive control , actin staining as loading control . The 72kD band correlates with harmonin _ a isoforms while higher MW indicates putative dimer formation . Middle panel : Immunofluorescence reveals absence of harmonin in the retina of the PBS - injected control eye and harmonin abundance ( green ) in the retina of the AAV - treated eye . ( Representative image from a single animal . Blue : DAPI , scale bar 25 µm ) . Right panel : Photopic gfERG reveal increased a - and b - wave response to single flash and flicker stimulation in the AAV - treated ( red ) , compared to the sham - treated ( black ) eyes of two USH1C pigs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Efficacy of AAV gene therapy was examined in vivo by sub-retinal injection of Anc80 virus capsid expressing harmonin_a1 under a ubiquitous promoter into a USH1C pig (see also Figure EV5). Left panel: Sub-retinal injection reconstituted harmonin expression in the AAV-treated eye but not in the sham-treated eye (3 TR). Protein extract from a WT retina served as positive control, actin staining as loading control. The 72kD band correlates with harmonin_a isoforms while higher MW indicates putative dimer formation. Middle panel: Immunofluorescence reveals absence of harmonin in the retina of the PBS-injected control eye and harmonin abundance (green) in the retina of the AAV-treated eye. (Representative image from a single animal. Blue: DAPI, scale bar 25 µm). Right panel: Photopic gfERG reveal increased a- and b-wave response to single flash and flicker stimulation in the AAV-treated (red), compared to the sham-treated (black) eyes of two USH1C pigs.


A - H ) slices through electron cryotomograms of H . gracilis cells ( left panels ) , central slices through subtomogram averages ( STA , middle panels ) and schematic representations ( right panels ) representing the various assembly stages observed . White arrow in ( A ) points to the MS - complex ; green arrows point to the novel 53 - nm - wide cytoplasmic ring . Scale bars in ( H ) are 50 nm for the cryotomogram slice and 20 nm for the STA and apply to all panels . Empty panels represent stages where not enough particles were found to produce a STA . OM = outer membrane , IM = inner membrane .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) slices through electron cryotomograms of H. gracilis cells (left panels), central slices through subtomogram averages (STA, middle panels) and schematic representations (right panels) representing the various assembly stages observed. White arrow in (A) points to the MS-complex; green arrows point to the novel 53-nm-wide cytoplasmic ring. Scale bars in (H) are 50 nm for the cryotomogram slice and 20 nm for the STA and apply to all panels. Empty panels represent stages where not enough particles were found to produce a STA. OM=outer membrane, IM=inner membrane.


A - D ) slices through electron cryotomograms of H . pylori ( left panels ) indicating the presence of various flagellar intermediates . Middle panels show central slices through STAs . Right panels show schematic representations . Question marks in the schematic in ( C ) indicate that it is unclear whether the densities surrounding the upper part of the C - ring are the same as the novel cytoplasmic rings seen in the MS - complex . Empty panels indicate that there were not enough examples to produce a STA .

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

A-D) slices through electron cryotomograms of H. pylori (left panels) indicating the presence of various flagellar intermediates. Middle panels show central slices through STAs. Right panels show schematic representations. Question marks in the schematic in (C) indicate that it is unclear whether the densities surrounding the upper part of the C-ring are the same as the novel cytoplasmic rings seen in the MS-complex. Empty panels indicate that there were not enough examples to produce a STA.


E & F ) cryotomogram slices , STAs , and schematics of H . pylori fliP * cells showing the MS - complex ( E ) and the C - complex ( F ) . The two novel cytoplasmic rings are highlighted with light and dark green arrows in the STA panel . White arrows in ( A - F ) point to the particles .

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

E & F) cryotomogram slices, STAs, and schematics of H. pylori fliP* cells showing the MS-complex (E) and the C-complex (F). The two novel cytoplasmic rings are highlighted with light and dark green arrows in the STA panel. White arrows in (A-F) point to the particles.


G & H ) central slices through the STAs of the MS - complexes of the motile H . pylori ( G ) and H . pylori FliP * mutant ( H ) with the diameters of their various constituent rings indicated .

O O O O O O O O O O O O O O O O O O O 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 & H) central slices through the STAs of the MS-complexes of the motile H. pylori (G)and H. pylori FliP* mutant (H) with the diameters of their various constituent rings indicated.


A - G ) Slices through electron cryotomograms ( left panels ) , central slices of subtomogram averages ( middle panels ) , and schematic representations ( right panels ) of the MS - and C - complexes ( white arrows on the slices ) of different mutants in H . pylori fliP * . Light and dark green arrows in ( A and D ) point to the ~ 53 - nm and ~ 67 - nm cytoplasmic rings . Scale bars for cryotomogram slices are 50 nm , and 20 nm for STAs . Empty panels indicate that there were not enough examples to produce a STA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Slices through electron cryotomograms (left panels), central slices of subtomogram averages (middle panels), and schematic representations (right panels) of the MS- and C-complexes (white arrows on the slices) of different mutants in H. pylori fliP*. Light and dark green arrows in (A and D) point to the ~53-nm and ~67-nm cytoplasmic rings. Scale bars for cryotomogram slices are 50 nm, and 20 nm for STAs. Empty panels indicate that there were not enough examples to produce a STA.


Treatment of HeLa cells with DFP ( 1mM for 24 h ) reduces the conjugation of SUMO - 2 / 3 ( n = 5 , biological replicates ; * * , p < 0 . 01 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) does not change global SUMO - 1 - ylation ( n = 5 , biological replicates ; p > 0 . 05 ; N . S . , not statistically significant ; paired t - test ) .

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 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

Treatment of HeLa cells with DFP (1mM for 24 h) reduces the conjugation of SUMO-2/3 (n=5, biological replicates; **, p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change global SUMO-1-ylation (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).


Treatment of HeLa cells with DFP ( 1mM for 24 h ) leads to increased levels of SENP3 ( n = 8 , biological replicates ; * * p < 0 . 01 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) does not change levels of SENP5 ( n = 5 , biological replicates ; p > 0 . 05 ; N . S . , not statistically significant ; paired t - test ) .

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 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

Treatment of HeLa cells with DFP (1mM for 24 h) leads to increased levels of SENP3 (n=8, biological replicates; **p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change levels of SENP5 (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).


DFP treatment increases SENP3 stability . DFP ( 1mM for 24 h ) - treated HeLa cells were treated with cycloheximide ( CHX ; 50μg / ml ) for the indicated durations in a chase experiment . Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated .

B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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

DFP treatment increases SENP3 stability. DFP (1mM for 24 h)-treated HeLa cells were treated with cycloheximide (CHX; 50μg/ml) for the indicated durations in a chase experiment. Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated.


Treatment of HeLa cells with DFP ( 1mM for 24 h ) decreases levels of CHIP ( n = 8 , biological replicates ; * * * p < 0 . 001 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) leads to decreased levels of CHIP mRNA ( n = 3 , biological replicates ; * p < 0 . 05 ; unpaired t - test ) .

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-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

Treatment of HeLa cells with DFP (1mM for 24 h) decreases levels of CHIP (n=8, biological replicates; ***p<0.001; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) leads to decreased levels of CHIP mRNA (n=3, biological replicates; *p<0.05; unpaired t-test).


Treatment of DFP ( 1mM for 24 h ) - treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP . Histogram shows relative levels of cellular CHIP upon four different conditions ( n = 3 , biological replicates ; * * * * p < 0 . 0001 ; * * * p < 0 . 001 ; repeated measures analysis of variance followed by Tukey ' s multiple comparisons test ) .

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 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

Treatment of DFP (1mM for 24 h)-treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP. Histogram shows relative levels of cellular CHIP upon four different conditions (n=3, biological replicates; ****p<0.0001; *** p<0.001; repeated measures analysis of variance followed by Tukey's multiple comparisons test).


CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells . Nsi or CHIPi ( Nsi , non - specific siRNA ; CHIPi , CHIP siRNA ; concentration , 20 nM ) was transfected into HeLa cells for 48 h . Lysate samples were blotted as indicated . CHIP knockdown prevents the DFP - induced increase in SENP3 levels . Nsi , SENP3i or CHIPi ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ; CHIPi , CHIP siRNA ; siRNA concentration , 20nM ) was transfected into HeLa cells for 48 h . Two days post - transfection the cells were treated with DFP for a further 24 h .

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

CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells. Nsi or CHIPi (Nsi, non-specific siRNA; CHIPi, CHIP siRNA; concentration, 20 nM) was transfected into HeLa cells for 48 h. Lysate samples were blotted as indicated.   CHIP knockdown prevents the DFP-induced increase in SENP3 levels. Nsi, SENP3i or CHIPi (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; CHIPi, CHIP siRNA; siRNA concentration, 20nM) was transfected into HeLa cells for 48 h. Two days post-transfection the cells were treated with DFP for a further 24 h.


SENP3 knockdown abolishes the DFP - induced increase in LC3 - II in HeLa cells . Nsi or SENP3i ( I ) was transfected into HeLa cells . Two days post - transfection the cells were treated with DFP for a further 24 h . Whole cell lysate samples were blotted as indicated ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ; siRNA concentration , 20nM ; DFP , 1mM for 24 h ; values normalized to the control value ; n = 3 , biological replicates ; * * * , p < 0 . 001 ; N . S . , not significant , p > 0 . 05 ; paired t - test ) .

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 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 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

SENP3 knockdown abolishes the DFP-induced increase in LC3-II in HeLa cells. Nsi or SENP3i (I) was transfected into HeLa cells. Two days post-transfection the cells were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; siRNA concentration, 20nM; DFP, 1mM for 24 h; values normalized to the control value; n=3, biological replicates; ***, p<0.001; N.S., not significant, p>0.05; paired t-test).


Nsi or SENP3i ( II ) was transfected into HeLa cells with stable expression of mito - Keima grown on 6 - well plates . Two days post - transfection the cells were treated with DFP ( 1mM ; 24 h ) . Cells transfected with Nsi and treated with CQ ( 50μM ; 24 h ) in the absence or presence of DFP ( 1mM ; 24 h ) were included as a negative control for mitophagy induction . The images show overlaid fluorescence and transmitted light images of unfixed , live cells obtained using an EVOS - fl inverted LED fluorescence microscope ( Red : mitophagic puncta ; Scale bar , 100 μm ) . Histogram shows the percentages of mitophagic cells under 6 different conditions ( n = 3 , biological replicates ; * * * * , p < 0 . 0001 ; Ordinary one - way ANOVA followed by Tukey ' s multiple comparisons test ) .

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

Nsi or SENP3i (II) was transfected into HeLa cells with stable expression of mito-Keima grown on 6-well plates. Two days post-transfection the cells were treated with DFP (1mM; 24 h). Cells transfected with Nsi and treated with CQ (50μM; 24 h) in the absence or presence of DFP (1mM; 24 h) were included as a negative control for mitophagy induction. The images show overlaid fluorescence and transmitted light images of unfixed, live cells obtained using an EVOS-fl inverted LED fluorescence microscope (Red: mitophagic puncta; Scale bar, 100 μm). Histogram shows the percentages of mitophagic cells under 6 different conditions (n=3, biological replicates; ****, p<0.0001; Ordinary one-way ANOVA followed by Tukey's multiple comparisons test).