( D ) Quantitative data . Bars depict the percentages of cells exhibiting the accumulation of RFP - LC3 in puncta ( RFP - LC3vac ) . ( B and D ) Means ± SEM ; n = 3 ; * , P < 0 . 05 .

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

(D) Quantitative data. Bars depict the percentages of cells exhibiting the accumulation of RFP-LC3 in puncta (RFP-LC3vac). (B and D) Means ± SEM; n = 3; *, P < 0.05.


( E ) Representative immunoblots for 24 - h endogenous LC3 lipidation . The asterisk stands for a nonspecific band . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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

(E) Representative immunoblots for 24-h endogenous LC3 lipidation. The asterisk stands for a nonspecific band. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo . ( A ) Human colorectal carcinoma HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and then treated with either vehicle ( Co , control ) or the indicated dose of resveratrol ( Resv ) or spermidine ( Spd ) , alone or in combination , for 2 h . ( top ) Quantitative data . Bars depict the percentages ( means ± SD ; n = 3 ; * * , P < 0 . 05 ) of cells showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) . ( bottom ) Representative immunoblots showing endogenous LC3 lipidation .

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

Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo. (A) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle (Co, control) or the indicated dose of resveratrol (Resv) or spermidine (Spd), alone or in combination, for 2 h. (top) Quantitative data. Bars depict the percentages (means ± SD; n = 3; **, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). (bottom) Representative immunoblots showing endogenous LC3 lipidation.


B ) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 ( BafA1 ) . ( C - E ) Transgenic C57BL / 6 mice expressing a GFP - LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations . 3 h later , mice were killed , and tissues were processed for immunofluorescence microscopy determinations of GFP - LC3vac .

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

B) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 (BafA1). (C-E) Transgenic C57BL/6 mice expressing a GFP-LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations. 3 h later, mice were killed, and tissues were processed for immunofluorescence microscopy determinations of GFP-LC3vac.


( D ) Quantitative results . Bars represent the percentages ( means ± SEM ; n = 3 ; * , P < 0 . 05 ; and * * , P < 0 . 01 as compared with the same tissue from untreated animals ) of cells exhibiting GFP - LC3vac .

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

(D) Quantitative results. Bars represent the percentages (means ± SEM; n = 3; *, P < 0.05; and **, P < 0.01 as compared with the same tissue from untreated animals) of cells exhibiting GFP-LC3vac.


( E ) Representative immunoblots showing endogenous LC3 lipidation and p62 protein content in WT C57BL / 6 mouse tissues . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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

(E) Representative immunoblots showing endogenous LC3 lipidation and p62 protein content in WT C57BL/6 mouse tissues. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


A ) Correlations of mean fold - changes of the proteins changing PD - dependently in the Columbia and LCC cohorts . Only proteins quantified in both cohorts are shown ( n = 330 ) . Proteins overlapping between the two cohorts are labeled with their name .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlations of mean fold-changes of the proteins changing PD-dependently in the Columbia and LCC cohorts. Only proteins quantified in both cohorts are shown (n=330). Proteins overlapping between the two cohorts are labeled with their name.


B ) Mean fold - changes for each of the 33 proteins that were LRRK2 - dependently regulated in both cohorts using a pairwise t - test comparing the four subgroups ( HC , NMC , iPD and LRRK2 PD ) .

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

B) Mean fold-changes for each of the 33 proteins that were LRRK2-dependently regulated in both cohorts using a pairwise t-test comparing the four subgroups (HC, NMC, iPD and LRRK2 PD).


A ) Pearson correlation scores and associated p - values [ - log10 ] of all protein intensities with the MoCA total score . Either all PD patients ( left ) , iPD patients ( middle ) or LRRK2 PD patients ( right ) were included in the analysis . Significantly correlated proteins with an FDR of 5 % after Benjamini - Hochberg correction are highlighted in 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 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

A) Pearson correlation scores and associated p-values [-log10] of all protein intensities with the MoCA total score. Either all PD patients (left), iPD patients (middle) or LRRK2 PD patients (right) were included in the analysis. Significantly correlated proteins with an FDR of 5% after Benjamini-Hochberg correction are highlighted in red.


B ) Pearson correlation scores and associated p - values [ - log10 ] of all protein intensities with the UPDRS - III score . Either all PD patients ( left ) , iPD patients ( middle ) or LRRK2 PD patients ( right ) were included . Significantly correlated proteins with an FDR of 5 % after Benjamini - Hochberg correction are highlighted in 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Pearson correlation scores and associated p-values [-log10] of all protein intensities with the UPDRS-III score. Either all PD patients (left), iPD patients (middle) or LRRK2 PD patients (right) were included. Significantly correlated proteins with an FDR of 5% after Benjamini-Hochberg correction are highlighted in red.


( a , b ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP ( a ) . The high and low 20 % were sorted ( a ) ,

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

(a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a),


( a , b ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP ( a ) . The high and low 20 % were sorted ( a ) , re - plated and treated with lysosomal protease inhibitors pepstatin and E - 64d for 1 h ; lysates were then immunoblotted for the indicated proteins ( b ) . ( c ) Densitometry of LC3 - II and p62 western blots ( normalized to actin and hour 0 , mean ± s . e . m . , n = 3 blots from 2 independent experiments , * P = 0 . 051 , * * P = 0 . 0091 ) .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a), re-plated and treated with lysosomal protease inhibitors pepstatin and E-64d for 1 h; lysates were then immunoblotted for the indicated proteins (b). (c) Densitometry of LC3-II and p62 western blots (normalized to actin and hour 0, mean ± s.e.m., n = 3 blots from 2 independent experiments, *P = 0.051, **P = 0.0091).


( d ) ; autophagic LC3 puncta were assessed by quantitative

(d); autophagic LC3 puncta were assessed by quantitative microscopy


( d ) ; autophagic LC3 puncta were assessed by quantitative microscopy ( e ; punctate area per cell , mean ± s . e . m . , n = 50 fields , * P = 0 . 010 , * * P = 0 . 053 ) .

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

(d); autophagic LC3 puncta were assessed by quantitative microscopy (e; punctate area per cell, mean ± s.e.m., n = 50 fields, *P = 0.010, **P = 0.053).


f ) Electron micrographs of HeLa mCherry - GFP - LC3 cells sorted for autophagic flux as in a . Yellow arrows denote autophagosomes ; red arrows indicate autolysosomes . ( g ) Quantification of autophagosomes and autolysomes from electron micrographs ( mean ± s . e . m . , n = 50 fields ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

f) Electron micrographs of HeLa mCherry-GFP-LC3 cells sorted for autophagic flux as in a. Yellow arrows denote autophagosomes; red arrows indicate autolysosomes. (g) Quantification of autophagosomes and autolysomes from electron micrographs (mean ± s.e.m., n = 50 fields). Uncropped images of blots are shown in Supplementary Fig. 6.


a - c ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP . Cells were then re - plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points ( median ratio of mCherry / GFP fluorescence ± s . e . m . , n = 3 wells ) . Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies ( b ) . Representative flow cytometry histograms for cells at 0 h and 24 h after sorting ( c ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP. Cells were then re-plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points (median ratio of mCherry/GFP fluorescence ± s.e.m., n = 3 wells). Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies (b). Representative flow cytometry histograms for cells at 0 h and 24 h after sorting (c).


( d ) BJAB mCherry - GFP - LC3 cells were sorted for autophagic flux as in a ( top and bottom 20 % ) . Following treatment with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) , cell viability was determined by MTS assay ( a tetrazolium reduction - based metabolic measurement ) at 24 h ( percentage of untreated control , mean ± s . e . m . , n = 3 wells , * P = 2 . 3 × 10 − 4 , * * P = 0 . 0036 ) .

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

(d) BJAB mCherry-GFP-LC3 cells were sorted for autophagic flux as in a (top and bottom 20%). Following treatment with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1), cell viability was determined by MTS assay (a tetrazolium reduction-based metabolic measurement) at 24 h (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 2.3×10−4, **P = 0.0036).


( e ) Long - term growth of BJAB mCherry - GFP - LC3 cells sorted for autophagic flux followed by treatment with Fas ligand ( 4 ng ml − 1 ) or TRAIL ( 15 ng ml − 1 ) for 24 h . Cells were then re - plated and allowed to recover for 5 ( Fas ligand ) or 6 ( TRAIL ) days then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 012 ) .

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

(e) Long-term growth of BJAB mCherry-GFP-LC3 cells sorted for autophagic flux followed by treatment with Fas ligand (4 ng ml−1) or TRAIL (15 ng ml−1) for 24 h. Cells were then re-plated and allowed to recover for 5 (Fas ligand) or 6 (TRAIL) days then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.012).


( f ) BJAB mCherry - GFP - LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI ( mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) .

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

(f) BJAB mCherry-GFP-LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI (mean ± s.e.m., n = 3 wells, *P = 0.0046).


( g ) Cells sorted as in a , were re - plated and , starting at the indicated times following sorting , treated with Fas ligand ( 4 ng ml − 1 ) for 24 h ; cell viability was then determined by MTS ( percentage of untreated control , mean ± s . e . m . , n = 3 wells ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

O 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

(g) Cells sorted as in a, were re-plated and, starting at the indicated times following sorting, treated with Fas ligand (4 ng ml−1) for 24 h; cell viability was then determined by MTS (percentage of untreated control, mean ± s.e.m., n = 3 wells). Uncropped images of blots are shown in Supplementary Fig. 6.


( a ) BJAB and Jurkat cells were treated with vehicle or chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by Fas ligand ( 1 . 5 ng ml − 1 ) . Cell viability was determined by MTS 24 h later ( percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 0 . 0058 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BJAB and Jurkat cells were treated with vehicle or chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (1.5 ng ml−1). Cell viability was determined by MTS 24 h later (percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 0.0058).


( b ) Immunoblots of cells in a , c probed for the indicated antibodies . CQ ,

(b) Immunoblots of cells in a,c probed for the indicated antibodies. CQ, chloroquine


( c ) BJAB and Jurkat cells were treated with chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by treatment with Fas ligand ( 4 ng ml − 1 ) for 24 h ( same technical replicate as a ) . Cells were then re - plated at low density in growth media and allowed to recover for 5 days , and then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0024 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BJAB and Jurkat cells were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by treatment with Fas ligand (4 ng ml−1) for 24 h (same technical replicate as a). Cells were then re-plated at low density in growth media and allowed to recover for 5 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0024).


( d ) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs , followed by 3 days of puromycin selection . Selected cells were plated and treated with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) for 24 h and viability was assessed by MTS ( percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 , * P = 1 . 7 × 10 − 4 , * * P = 0 . 024 ) .

O O O O O O O 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 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

(d) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs, followed by 3 days of puromycin selection. Selected cells were plated and treated with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1) for 24 h and viability was assessed by MTS (percentage of control (no ligand), mean ± s.e.m., n = 3, *P = 1.7×10−4, **P = 0.024).


( e ) Immunoblots for Atg5 , Atg7 and p62 confirm protein depletion and autophagy inhibition in d , f .

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

(e) Immunoblots for Atg5, Atg7 and p62 confirm protein depletion and autophagy inhibition in d,f.


( f ) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand ( 15 ng ml − 1 ) or TRAIL ( 12 . 5 ng ml − 1 ) for 24 h ( same technical replicate as d ) . Cells were then re - plated at low density and allowed to recover for 5 - 6 days , and then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0089 ) . Atg7 knockdown data were not included for long - term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(f) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand (15 ng ml−1) or TRAIL (12.5 ng ml−1) for 24 h (same technical replicate as d). Cells were then re-plated at low density and allowed to recover for 5-6 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0089). Atg7 knockdown data were not included for long-term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment. Uncropped images of blots are shown in Supplementary Fig. 6.


( a , b ) BJAB and Jurkat cells stably expressing mCherry - GFP - LC3 were flow sorted for high and low autophagic flux , treated with Fas ligand ( 1 . 5 ng ml − 1 ) for 24 h and viability was assessed by MTS ( a ; percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) . ( b ) Immunoblots following sorting of the samples in a .

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

(a,b) BJAB and Jurkat cells stably expressing mCherry-GFP-LC3 were flow sorted for high and low autophagic flux, treated with Fas ligand (1.5 ng ml−1) for 24 h and viability was assessed by MTS (a; percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0046). (b) Immunoblots following sorting of the samples in a.


( c , d ) The indicated cell lines were treated with chloroquine ( BJAB , CEM 20 μM ; SKW6 . 4 , 25 μM ; Jurkat , 10 μM ) for 16 h , followed by Fas ligand ( BJAB , 12 . 5 ng ml − 1 ; SKW6 . 4 , 50 ng ml − 1 ; Jurkat , 0 . 4 ng ml − 1 ; CEM , 40 ng ml − 1 ) . Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies ( c ) .

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

(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c).


( c , d ) The indicated cell lines were treated with chloroquine ( BJAB , CEM 20 μM ; SKW6 . 4 , 25 μM ; Jurkat , 10 μM ) for 16 h , followed by Fas ligand ( BJAB , 12 . 5 ng ml − 1 ; SKW6 . 4 , 50 ng ml − 1 ; Jurkat , 0 . 4 ng ml − 1 ; CEM , 40 ng ml − 1 ) . Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies ( c ) . Cell viability was assessed by MTS 24 h following Fas ligand treatment ( d ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 1 . 6 × 10 − 4 ) .

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

(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c). Cell viability was assessed by MTS 24 h following Fas ligand treatment (d; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 1.6×10−4).


( e , f ) BJAB and Jurkat cells were transduced with control , Atg5 , Atg7 or Vps34 shRNA lentiviruses , followed by three days of puromycin selection . Cells were then treated with Fas ligand ( 1 . 25 ng ml − 1 ) or TRAIL ( 1 . 25 ng ml − 1 ) for 24 h and viability was assessed by MTS ( e ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 1 . 4 × 10 − 6 , * * P = 2 . 3 × 10 − 5 , * * * P = 3 . 9 × 10 − 5 , § P = 9 . 2 × 10 − 6 , § § P = 3 . 4 × 10 − 6 , § § § P = 1 . 7 × 10 − 5 ) . Immunoblots demonstrate Atg5 , Atg7 and Vps34 knockdown , autophagy inhibition and altered Fap - 1 levels . The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other ; the Fap - 1 blot is not separated to demonstrate the lack of Fap - 1 protein in Type II Jurkat cells ( f ) . Fap - 1 blots ( b , c , f ) were run on separate gels owing to the quantity of protein required for detection ( see Methods ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(e,f) BJAB and Jurkat cells were transduced with control, Atg5, Atg7 or Vps34 shRNA lentiviruses, followed by three days of puromycin selection. Cells were then treated with Fas ligand (1.25 ng ml−1) or TRAIL (1.25 ng ml−1) for 24 h and viability was assessed by MTS (e; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 1.4×10−6, **P = 2.3×10−5, ***P = 3.9×10−5, §P = 9.2×10−6, §§P = 3.4×10−6, §§§P = 1.7×10−5). Immunoblots demonstrate Atg5, Atg7 and Vps34 knockdown, autophagy inhibition and altered Fap-1 levels. The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other; the Fap-1 blot is not separated to demonstrate the lack of Fap-1 protein in Type II Jurkat cells (f). Fap-1 blots (b,c,f) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.


( a , b ) BJAB and Jurkat cells expressing the indicated Fap - 1 wild - type ( WT ) and catalytically inactive ( ΔCD ) constructs were treated with chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by Fas ligand ( BJAB , 1 . 5 ng ml − 1 ; Jurkat 15 ng ml − 1 ) for 24 h . Cell viability was determined by MTS ( a ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 2 . 4 × 10 − 5 , * * * P = 0 . 0024 ) . Cell lysates were blotted and probed with the indicated antibodies ( b ) .

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

(a,b) BJAB and Jurkat cells expressing the indicated Fap-1 wild-type (WT) and catalytically inactive (ΔCD) constructs were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (BJAB, 1.5 ng ml−1; Jurkat 15 ng ml−1) for 24 h. Cell viability was determined by MTS (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 2.4×10−5, ***P = 0.0024). Cell lysates were blotted and probed with the indicated antibodies (b).


c - e ) BJAB cells expressing control or Fap - 1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand ( 12 . 5 ng ml − 1 ) or TRAIL ( 25 ng ml − 1 ) for 24 h ; cell viability was then determined by MTS ( c ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 4 . 8 × 10 − 6 , * * P = 8 . 1 × 10 − 5 , * * * P = 8 . 0 × 10 − 4 , § P = 0 . 013 , NS P > 0 . 05 ) .

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 B-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 O O O O O

c-e) BJAB cells expressing control or Fap-1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand (12.5 ng ml−1) or TRAIL (25 ng ml−1) for 24 h; cell viability was then determined by MTS (c; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 4.8×10−6, **P = 8.1×10−5, ***P = 8.0×10−4, §P = 0.013, NS P>0.05).


( d ) Immunoblots of lysates collected following chloroquine treatment . Long - term growth was assessed in cells from c by re - plating them at low density and allowing them to recover for 5 days , followed by viability assay ( percentage of untreated control , mean ± s . e . m . , n = 3 wells , * P = 0 . 043 ; e ) . Fap - 1 blots ( b , d ) were run on separate gels owing to the quantity of protein required for detection ( see Methods ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(d) Immunoblots of lysates collected following chloroquine treatment. Long-term growth was assessed in cells from c by re-plating them at low density and allowing them to recover for 5 days, followed by viability assay (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 0.043; e). Fap-1 blots (b,d) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.


a , b ) Cell viability was determined by MTS assay following Fas ligand ( 4 ng ml − 1 ) treatment in BJAB cells expressing control or p62 lentiviral shRNAs ( a ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 3 . 2 × 10 − 5 , * * P = 1 . 8 × 10 − 6 ) . Immunoblots confirm p62 depletion and Fap - 1 levels ( b ) .

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

a,b) Cell viability was determined by MTS assay following Fas ligand (4 ng ml−1) treatment in BJAB cells expressing control or p62 lentiviral shRNAs (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 3.2×10−5, **P = 1.8×10−6). Immunoblots confirm p62 depletion and Fap-1 levels (b).


( c ) Co - immunoprecipitation of endogenous p62 and Fap - 1 in BJAB cells treated with 20 μM chloroquine for 16 h , followed by treatment with Fas ligand ( 50 ng ml − 1 ) for 2 h at 4 ° C .

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

(c) Co-immunoprecipitation of endogenous p62 and Fap-1 in BJAB cells treated with 20 μM chloroquine for 16 h, followed by treatment with Fas ligand (50 ng ml−1) for 2 h at 4°C.


HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting . ( A ) Filter ‐ trap analysis of MG132 ‐ induced , SDS ‐ insoluble ubiquitinated aggregates generated in wild ‐ type ( WT ) , HDAC6 KO , and KO MEFs reconstituted with different HDAC6 constructs as indicated . The signal intensity from the ubiquitinimmunoblot ( bottom panel ) was quantified and presented as the average of the means from three independent experiments along with the standard deviation ( top ) . Note the significant accumulation of ubiquitin ‐ positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6 ‐ CD and ΔBUZ mutants . WT , HDAC6 KO , and HDAC6 KO MEFs stably expressing human HDAC6 ( hHDAC6 WT ) , HDAC6 CD ( catalytically inactive mutant ) , or HDAC6 ΔBUZ ( ubiquitin ‐ binding ‐ deficient mutant ) were analysed for the level of HDAC6 using an anti ‐ human HDAC6 antibody . Mouse endogenous HDAC6 ( mHDAC6 ) and actin levels were determined by each corresponding antibody .

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

HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting. (A) Filter‐trap analysis of MG132‐induced, SDS‐insoluble ubiquitinated aggregates generated in wild‐type (WT), HDAC6 KO, and KO MEFs reconstituted with different HDAC6 constructs as indicated. The signal intensity from the ubiquitinimmunoblot (bottom panel) was quantified and presented as the average of the means from three independent experiments along with the standard deviation (top). Note the significant accumulation of ubiquitin‐positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6‐CD and ΔBUZ mutants. WT, HDAC6 KO, and HDAC6 KO MEFs stably expressing human HDAC6 (hHDAC6 WT), HDAC6 CD (catalytically inactive mutant), or HDAC6 ΔBUZ (ubiquitin‐binding‐deficient mutant) were analysed for the level of HDAC6 using an anti‐human HDAC6 antibody. Mouse endogenous HDAC6 (mHDAC6) and actin levels were determined by each corresponding antibody.


( B ) WT and HDAC6 KO MEFs were treated with MG132 and subjected to Western blot analysis for LC3 , actin , and HDAC6 .

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

(B) WT and HDAC6 KO MEFs were treated with MG132 and subjected to Western blot analysis for LC3, actin, and HDAC6.


( C ) Cells were treated with MG132 as described under Materials and methods and immunostained with antibodies to ubiquitin ( green ) and LC3 ( red ) . Arrows indicate ubiquitin ‐ positive aggregates that colocalize with LC3 ‐ positive autophagosomes . Scale bar , 10 μm .

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

(C) Cells were treated with MG132 as described under Materials and methods and immunostained with antibodies to ubiquitin (green) and LC3 (red). Arrows indicate ubiquitin‐positive aggregates that colocalize with LC3‐positive autophagosomes. Scale bar, 10 μm.


( D ) WT and ATG5 KO MEFs are treated with 2 . 5 μM MG132 for 1 day and incubated with normal growth media for 18 h . MEFs are immunostained with anti ‐ LC3 ( red ) and anti ‐ ubiquitin antibody ( green ) .

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

(D) WT and ATG5 KO MEFs are treated with 2.5 μM MG132 for 1 day and incubated with normal growth media for 18 h. MEFs are immunostained with anti‐LC3 (red) and anti‐ubiquitin antibody (green).


( A ) Wild ‐ type and HDAC6 KO MEFs were transfected with pcDNA , pcDNA ‐ HDAC6WT , HDAC6CD , or HDAC6ΔBuz , along with mCherry ‐ GFP ‐ LC3 as indicated . Yellow signals indicate non ‐ acidic autophagosomes and red signals indicate acidic autophagolysosomes . Scale bar , 10 μm .

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

(A) Wild‐type and HDAC6 KO MEFs were transfected with pcDNA, pcDNA‐HDAC6WT, HDAC6CD, or HDAC6ΔBuz, along with mCherry‐GFP‐LC3 as indicated. Yellow signals indicate non‐acidic autophagosomes and red signals indicate acidic autophagolysosomes. Scale bar, 10 μm.


( B ) The total number of yellow vesicles was quantified from three independent experiments ( > 12 cells each ) and presented as the percentage of total mCherry ‐ GFP ‐ LC3 dots ( red plus yellow ) along with the standard deviation .

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

(B) The total number of yellow vesicles was quantified from three independent experiments (>12 cells each) and presented as the percentage of total mCherry‐GFP‐LC3 dots (red plus yellow) along with the standard deviation.


( C ) In vitro fusion assays . Autophagosomes ( APGs ) and lysosomes ( Lys ) purified from wild type and HDAC6 − / − MEFs were subjected to heterotypic and homotypic in vitro fusion assays ( representative fields are shown in Supplementary Figure S3 ) . Values are means + s . e . of the percentages of fusion from three independent experiments ( more than 10 images per each experiment ) .

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

(C)In vitro fusion assays. Autophagosomes (APGs) and lysosomes (Lys) purified from wild type and HDAC6−/− MEFs were subjected to heterotypic and homotypic in vitro fusion assays (representative fields are shown in Supplementary Figure S3). Values are means+s.e. of the percentages of fusion from three independent experiments (more than 10 images per each experiment).


( D ) EM images of wild ‐ type and HDAC6 KO MEFs in normal growth conditions . Yellow arrows , autophagosomes ; red arrows , autophagolysosomes ; green arrowheads , multilamellar bodies .

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

(D) EM images of wild‐type and HDAC6 KO MEFs in normal growth conditions. Yellow arrows, autophagosomes; red arrows, autophagolysosomes; green arrowheads, multilamellar bodies.


( E ) Quantification of autophagosomes and autophagolysosomes . The error bar represents the standard error .

(E) Quantification of autophagosomes and autophagolysosomes. The error bar represents the standard error.


( A ) Autophagosome - lysosome fusion is analysed in wild ‐ type and HDAC6 KO MEFs with or without starvation ( 6 h ) using mCherry ‐ GFP ‐ LC3 as described in Figure 2A .

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

(A) Autophagosome-lysosome fusion is analysed in wild‐type and HDAC6 KO MEFs with or without starvation (6 h) using mCherry‐GFP‐LC3 as described in Figure 2A.


( B ) Wild ‐ type and HDAC6 KO MEFs were cultured in Hank ' s solution for 3 h followed by immunoblotting with an antibody for LC3 , HDAC6 , and GAPDH .

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-MEASURED_VAR O B-MEASURED_VAR O O O O

(B) Wild‐type and HDAC6 KO MEFs were cultured in Hank's solution for 3 h followed by immunoblotting with an antibody for LC3, HDAC6, and GAPDH.


( C ) Long ‐ lived protein degradation in wild ‐ type and HDAC6 KO MEF cells . The degradation of [ 14C ] ‐ valine labelled long ‐ lived protein was measured in the presence or absence of 3 ‐ methyl adenine ( 3MA , inhibits the formation of autophagic vacuoles ) . The average of percentage degradation from three independent experiments is presented . The error bar represents the standard deviation .

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

(C) Long‐lived protein degradation in wild‐type and HDAC6 KO MEF cells. The degradation of [14C]‐valine labelled long‐lived protein was measured in the presence or absence of 3‐methyl adenine (3MA, inhibits the formation of autophagic vacuoles). The average of percentage degradation from three independent experiments is presented. The error bar represents the standard deviation.


( A ) Wild ‐ type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to Lamp ‐ 1 ( a lysosome marker , red ) and ubiquitin ( green ) as indicated . F ‐ actin was detected by phalloidin ( blue ) . The arrows indicate ubiquitin ‐ positive aggregates that are surrounded by F ‐ actin and Lamp ‐ 1 .

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

(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to Lamp‐1 (a lysosome marker, red) and ubiquitin (green) as indicated. F‐actin was detected by phalloidin (blue). The arrows indicate ubiquitin‐positive aggregates that are surrounded by F‐actin and Lamp‐1.


( B , C ) Wild ‐ type and HDAC6 KO MEFs were transfected with mCherry ‐ GFP ‐ LC3 , followed by treatment with LatA ( 100 nM ) or nocodazole ( 250 nM ) for 6 h and analysed as described in Figure 3A .

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

(B, C) Wild‐type and HDAC6 KO MEFs were transfected with mCherry‐GFP‐LC3, followed by treatment with LatA (100 nM) or nocodazole (250 nM) for 6 h and analysed as described in Figure 3A.


( D ) Autophagosomes ( APGs ) and lysosomes ( Lys ) isolated from fed mouse hepatocytes were treated or not with latrunculin ( LatA ) as indicated , extensively washed to remove traces of the inhibitor , and then labelled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin . The number of total fusion events / total number of vesicles for each condition was as follows : 176 / 880 ; 233 / 1110 ; 84 / 930 ; and 180 / 950 . The differences with untreated samples were significant at * * P0 . 01 .

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

(D) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed mouse hepatocytes were treated or not with latrunculin (LatA) as indicated, extensively washed to remove traces of the inhibitor, and then labelled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin. The number of total fusion events/total number of vesicles for each condition was as follows: 176/880; 233/1110; 84/930; and 180/950. The differences with untreated samples were significant at **P0.01.


( E ) Autophagosomes ( APGs ) and lysosomes ( Lys ) isolated from fed or starved mouse hepatocytes were treated or not with latrunculin ( LatA ) as labelled and subjected to in vitro fusion assay . The differences with untreated samples were significant at * P0 . 05 . The number of total fusion events / total number of vesicles for each condition was as follows : 169 / 1250 ; 87 / 972 ; 190 / 1120 ; and 175 / 1165 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed or starved mouse hepatocytes were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at *P0.05. The number of total fusion events/total number of vesicles for each condition was as follows: 169/1250; 87/972; 190/1120; and 175/1165.


( F ) Autophagosomes ( APGs ) and lysosomes ( Lys ) isolated from HDAC6 KO MEFs were treated or not with latrunculin ( LatA ) as indicated and subjected to in vitro fusion assay .

O O O O O O O O O O O O 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) Autophagosomes (APGs) and lysosomes (Lys) isolated from HDAC6 KO MEFs were treated or not with latrunculin (LatA) as indicated and subjected to in vitro fusion assay.


( A ) Biochemical characterization of autophagic compartments isolated from HDAC6 KO cells . Different subcellular fractions ( 75 μg protein ) isolated from the wild type ( WT ) were subjected to SDS - PAGE and immunoblotting for the indicated proteins . Hom , homogenate ; APG , autophagosomes ; APL , autophagolysosomes ; Lys , lysosomes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Biochemical characterization of autophagic compartments isolated from HDAC6 KO cells. Different subcellular fractions (75 μg protein) isolated from the wild type (WT) were subjected to SDS-PAGE and immunoblotting for the indicated proteins. Hom, homogenate; APG, autophagosomes; APL, autophagolysosomes; Lys, lysosomes.


( B ) Autophagosomes ( APGs ) and lysosomes ( Lys ) isolated from fed cells were treated or not with latrunculin ( LatA ) as labelled and subjected to in vitro fusion assay . The differences with untreated samples were significant at * * P0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed cells were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at **P0.01.


( C ) WT and ATG5 KO MEFs were treated with MG132 and immunostained with antibodies to ubiquitin ( green ) as indicated . F ‐ actin was detected by phalloidin ( red ) . The arrows indicated ubiquitin ‐ positive protein aggregates .

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

(C) WT and ATG5 KO MEFs were treated with MG132 and immunostained with antibodies to ubiquitin (green) as indicated. F‐actin was detected by phalloidin (red). The arrows indicated ubiquitin‐positive protein aggregates.


( A ) Wild ‐ type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies against cortactin ( red ) , ubiquitin ( green ) , and phalloidin for F ‐ actin ( blue ) as indicated . The arrows indicated ubiquitin ‐ positive aggregates that were colocalized with F ‐ actin and cortactin .

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

(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies against cortactin (red), ubiquitin (green), and phalloidin for F‐actin (blue) as indicated. The arrows indicated ubiquitin‐positive aggregates that were colocalized with F‐actin and cortactin.


( B ) Wild ‐ type MEFs were transfected with control or cortactin siRNA , treated with MG132 , and stained with antibodies for Lap ‐ 1 ( red , to label lysosome ) , or ubiquitin ( green ) and phalloidin for actin ( blue ) . Note that F ‐ actin staining at protein aggregates was lost , but lysosomes remained concentrated in cortactin knockdown cells ( arrow ) .

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

(B) Wild‐type MEFs were transfected with control or cortactin siRNA, treated with MG132, and stained with antibodies for Lap‐1 (red, to label lysosome), or ubiquitin (green) and phalloidin for actin (blue). Note that F‐actin staining at protein aggregates was lost, but lysosomes remained concentrated in cortactin knockdown cells (arrow).


( C ) Wild ‐ type MEFs were transfected with control or cortactin siRNA , treated MG132 2 . 5 μM for 18 h , and subjected to filter ‐ trap assay using a ubiquitin antibody . The knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody against cortactin and GAPDH in the right panel .

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

(C) Wild‐type MEFs were transfected with control or cortactin siRNA, treated MG132 2.5 μM for 18 h, and subjected to filter‐trap assay using a ubiquitin antibody. The knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody against cortactin and GAPDH in the right panel.


( D ) U2OS cells were transfected with control siRNA and cortactin siRNA . Autophagosome - lysosome fusion was analysed with or without starvation ( 6 h ) using the mCherry ‐ GFP ‐ LC3 reporter as described in Figure 3A .

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

(D) U2OS cells were transfected with control siRNA and cortactin siRNA. Autophagosome-lysosome fusion was analysed with or without starvation (6 h) using the mCherry‐GFP‐LC3 reporter as described in Figure 3A.


( E ) The mCherry ‐ GFP ‐ LC3 plasmid was cotransfected with wild type , 9KQ ( acetylation ‐ mimic ) , or 9KR ( deacetylation ‐ mimic ) cortactin ‐ expressing plasmids into wild ‐ type MEFs . Autophagosome - lysosome fusion was analysed as described in Figure 3A .

O O O O O O 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

(E) The mCherry‐GFP‐LC3 plasmid was cotransfected with wild type, 9KQ (acetylation‐mimic), or 9KR (deacetylation‐mimic) cortactin‐expressing plasmids into wild‐type MEFs. Autophagosome-lysosome fusion was analysed as described in Figure 3A.


( A ) HDAC6 KO mice accumulate ubiquitin ‐ positive protein aggregates in the brain . The hippocampus and cerebral cortex regions from 6 ‐ month ‐ old wild ‐ type and ubiquitin KO littermates were subjected to immunostaining with a ubiquitin antibody and counterstained with hematoxylin . The red arrows indicate ubiquitin ‐ positive neuritic aggregates and black arrows indicate cytoplasmic aggregates . These ubiquitin ‐ positive structures were rarely observed in control littermates . Scale bar , 50

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

(A) HDAC6 KO mice accumulate ubiquitin‐positive protein aggregates in the brain. The hippocampus and cerebral cortex regions from 6‐month‐old wild‐type and ubiquitin KO littermates were subjected to immunostaining with a ubiquitin antibody and counterstained with hematoxylin. The red arrows indicate ubiquitin‐positive neuritic aggregates and black arrows indicate cytoplasmic aggregates. These ubiquitin‐positive structures were rarely observed in control littermates. Scale bar, 50 μm


( B ) Apoptotic cell death in the cortex and hippocampus region of HDAC6 KO mice as determined by TUNEL staining . Apoptotic cells were only observed in HDAC6 KO mice . Scale bar , 100 μm .

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

(B) Apoptotic cell death in the cortex and hippocampus region of HDAC6 KO mice as determined by TUNEL staining. Apoptotic cells were only observed in HDAC6 KO mice. Scale bar, 100 μm.


( C ) HDAC6 depletion in the Drosophila eye leads to ubiquitin ‐ positive pathology . Immunostaining for ubiquitin ( green ) in frontal eye sections of 1 ‐ day ‐ old ( d1 ) and 30 ‐ day ‐ old ( d30 ) fly eyes . The eyes of HDAC6 ‐ depleted flies ( GMR : GAL4 / UAS ‐ HDAC6RNAi ) developed ubiquitin ‐ positive cytoplasmic inclusions that become more prominent at day 30 ( d30 ) . Blue , DAPI .

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

(C) HDAC6 depletion in the Drosophila eye leads to ubiquitin‐positive pathology. Immunostaining for ubiquitin (green) in frontal eye sections of 1‐day‐old (d1) and 30‐day‐old (d30) fly eyes. The eyes of HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) developed ubiquitin‐ positive cytoplasmic inclusions that become more prominent at day 30 (d30). Blue, DAPI.


( D ) Depletion of HDAC6 in the Drosophila eye leads to age ‐ dependent degeneration . Light micrographs ( left ) and corresponding Richardson ‐ stained frontal eye sections ( right ) of 1 ‐ day ‐ old and 30 ‐ day ‐ old fly eyes . The eyes of control flies ( GMR : GAL4 / + ) and HDAC6 ‐ depleted flies ( GMR : GAL4 / UAS ‐ HDAC6RNAi ) show normal highly organized ommatidial array at day 1 ; 30 ‐ day ‐ old control animals also show no defects , but 30 ‐ day ‐ old HDAC6 ‐ depleted flies show degeneration with disorganization of the ommatidial array and loss of normal eye architecture ( × 40 and × 80 ) .

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

(D) Depletion of HDAC6 in the Drosophila eye leads to age‐dependent degeneration. Light micrographs (left) and corresponding Richardson‐stained frontal eye sections (right) of 1‐day‐old and 30‐day‐old fly eyes. The eyes of control flies (GMR:GAL4/+) and HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) show normal highly organized ommatidial array at day 1; 30‐day‐old control animals also show no defects, but 30‐day‐old HDAC6‐depleted flies show degeneration with disorganization of the ommatidial array and loss of normal eye architecture ( × 40 and × 80).


( A , B ) The mRNA levels of aub ( A ) and vasa ( B ) were quantified upon the depletion ( RNAi ) of L ( 3 ) mbt , Lint - 1 , CoRest , E2F2 , Myb , Mip120 , and Mip130 , and were compared with those in normal OSCs ( control ) . Data represent the means ± SE ( n = 3 biological replicates ) . The p values were calculated with the t - test . * p : < 0 . 05 , * * p : < 0 . 01 . All t - tests were performed against samples with ⎾ symbol .

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

(A, B) The mRNA levels of aub (A) and vasa (B) were quantified upon the depletion (RNAi) of L(3)mbt, Lint-1, CoRest, E2F2, Myb, Mip120, and Mip130, and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05, **p: < 0.01. All t-tests were performed against samples with⎾ symbol.


( C ) Proteins immunoprecipitated with the anti - L ( 3 ) mbt antibody from the OSC lysates before ( control ) and after L ( 3 ) mbt knockdown ( KD ) were silver - stained . The bands corresponding to L ( 3 ) mbt - L and L ( 3 ) mbt - S are indicated .

(C) Proteins immunoprecipitated with the anti-L(3)mbt antibody from the OSC lysates before (control) and after L(3)mbt knockdown (KD) were silver-stained. The bands corresponding to L(3)mbt-L and L(3)mbt-S are indicated.


( F , G ) The mRNA levels of vasa ( F ) and aub ( G ) were quantified upon the depletion ( RNAi ) of L ( 3 ) mbt , Atac3 , Prod , CG2662 / Lint - O , and CG2199 , and were compared with those in normal OSCs ( control ) . Data represent the means ± SE ( n = 3 biological replicates ) . The p values were calculated with the t - test . * * p : < 0 . 01 . All t - tests were performed against samples with ⎾ symbol .

O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) The mRNA levels of vasa (F) and aub (G) were quantified upon the depletion (RNAi) of L(3)mbt, Atac3, Prod, CG2662/Lint-O, and CG2199, and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. **p: < 0.01. All t-tests were performed against samples with⎾ symbol.


( B ) Immunoprecipitation ( IP ) / western blotting shows that WT Lint - O , but not its ∆ SAM mutant co - immunoprecipitated with L ( 3 ) mbt from the OSC lysates . n . i . : non - immune IgG .

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

(B) Immunoprecipitation (IP)/western blotting shows that WT Lint-O, but not its ∆SAM mutant co-immunoprecipitated with L(3)mbt from the OSC lysates. n.i.: non-immune IgG.


( C ) Subcellular localization of WT Lint - O and its ∆ SAM mutant ( green ) . Both proteins were localized to the nuclei ( DAPI : blue ) . Scale bar : 5 μm .

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

(C) Subcellular localization of WT Lint-O and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.


( D ) IP / western blotting shows that WT L ( 3 ) mbt - L , but not its ∆ SAM mutant co - immunoprecipitated with Lint - O from the OSC lysates . n . i . : non - immune IgG .

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

(D) IP/western blotting shows that WT L(3)mbt-L, but not its ∆SAM mutant co-immunoprecipitated with Lint-O from the OSC lysates. n.i.: non-immune IgG.


( E ) Subcellular localization of WT L ( 3 ) mbt - L and its ∆ SAM mutant ( green ) . Both proteins were localized to the nuclei ( DAPI : blue ) . Scale bar : 5 μm .

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

(E) Subcellular localization of WT L(3)mbt-L and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.


( F , G ) The mRNA levels of vasa ( F ) and aub ( G ) were quantified upon ectopic expression of EGFP , as well as WT Lint - O and its ∆ SAM and 8CA mutants ( Fig EV3A ) , in Lint - O - lacking OSCs ( Lint - O RNAi ) and were compared with those in normal OSCs ( control ) . Data represent the means ± SE ( n = 3 biological replicates ) . The p values were calculated with the t - test . * p : < 0 . 05 . All t - tests were performed against samples with ⎾ symbol .

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

(F, G) The mRNA levels of vasa (F) and aub (G) were quantified upon ectopic expression of EGFP, as well as WT Lint-O and its ∆SAM and 8CA mutants (Fig EV3A), in Lint-O-lacking OSCs (Lint-O RNAi) and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05. All t-tests were performed against samples with⎾ symbol.


( H ) IP / western blotting shows that L ( 3 ) mbt co - immunoprecipitated with WT Lint - O and its 8CA mutant , but not with ∆ PHD mutant An asterisk shows background .

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

(H) IP/western blotting shows that L(3)mbt co-immunoprecipitated with WT Lint-O and its 8CA mutant, but not with ∆PHD mutant An asterisk shows background.


( I ) Subcellular localization of WT Lint - O and its 8CA and ∆ PHD mutants ( green ) . All proteins were localized to the nuclei ( DAPI : blue ) . Scale bar : 5 μm .

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

(I) Subcellular localization of WT Lint-O and its 8CA and ∆PHD mutants (green). All proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.


( J ) ChIP - qPCR shows that L ( 3 ) mbt binding to the vasa promoter was weakened after the loss of Lint - O . Data represent the means ± SE ( n = 3 biological replicates ) . The p values were calculated with the t - test . * * p : < 0 . 01 . All t - tests were performed against samples with ⎾ symbol .

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

(J) ChIP-qPCR shows that L(3)mbt binding to the vasa promoter was weakened after the loss of Lint-O. Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. **p: < 0.01. All t-tests were performed against samples with⎾ symbol.


( K ) Western blotting showing the amounts of L ( 3 ) mbt , Lint - O , and histone H3 ( H3 ) in normal ( control ) , L ( 3 ) mbt - depleted ( RNAi ) , and Lint - O - depleted ( RNAi ) OSCs .

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

(K) Western blotting showing the amounts of L(3)mbt, Lint-O, and histone H3 (H3) in normal (control), L(3)mbt-depleted (RNAi), and Lint-O-depleted (RNAi) OSCs.


( L ) The lint - O mRNA levels in normal ( control ) , L ( 3 ) mbt - depleted ( RNAi ) , and Lint - O - depleted ( RNAi ) OSCs are shown by fragments per kilobase million ( FPKM ) . Data represent the means ± SE ( n = 3 biological replicates ) . The p values were calculated with the t - test . * p : < 0 . 05 , * * p : < 0 . 01 . All t - tests were performed against samples with ⎾ symbol .

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

(L) The lint-O mRNA levels in normal (control), L(3)mbt-depleted (RNAi), and Lint-O-depleted (RNAi) OSCs are shown by fragments per kilobase million (FPKM). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05, **p: < 0.01. All t-tests were performed against samples with⎾ symbol.


( B ) RT - qPCR analysis shows the mRNA levels of lint - O in y w and Lint - OKO ovaries . Data are expressed as mean and error bars represent SD . n = 2 biological replicates .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O 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) RT-qPCR analysis shows the mRNA levels of lint-O in y w and Lint-OKO ovaries. Data are expressed as mean and error bars represent SD. n = 2 biological replicates.


( D ) Confocal images of y w and Lint - OKO ovaries immunostained for Vasa . Scale bar : 50 μm .

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

(D) Confocal images of y w and Lint-OKO ovaries immunostained for Vasa. Scale bar: 50 μm.


( E ) Confocal images of y w and Lint - OKO ovarioles immunostained for Vasa . Vasa was ectopically expressed in follicle cells ( white arrowheads ) . Scale bar : 50 μm .

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

(E) Confocal images of y w and Lint-OKO ovarioles immunostained for Vasa. Vasa was ectopically expressed in follicle cells (white arrowheads). Scale bar: 50 μm.


( A ) Western blotting shows that Vasa , Piwi , and Aub were ectopically expressed in Lint - OKO and L ( 3 ) mbtts1 larval brains at 29ºC . Anti - Lint - O , anti - L ( 3 ) mbt , anti - Vasa , andi - Piwi , anti - Aub , and anti - Tubulin antibodies were used .

(A) Western blotting shows that Vasa, Piwi, and Aub were ectopically expressed in Lint-OKO and L(3)mbtts1 larval brains at 29ºC. Anti-Lint-O, anti-L(3)mbt, anti-Vasa, andi-Piwi, anti-Aub, and anti-Tubulin antibodies were used.


( B ) Confocal images of y w , Lint - OKO , and L ( 3 ) mbtts1 immunostained for Vasa ( magenta ) , MIRA ( green ) , and ELAV ( green ) . Scale bar : 100 μm .

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

(B) Confocal images of y w, Lint-OKO, and L(3)mbtts1 immunostained for Vasa (magenta), MIRA (green), and ELAV (green). Scale bar: 100 μm.


( C ) Quantification of brain lobe volume for the following genotypes : Lint - OKO - / + ( n = 42 biological replicates ) , Lint - OKO - / - ( n = 36 biological replicates ) , and L ( 3 ) mbtts1 - / - ( n = 34 biological replicates ) . Boxplot central bands , upper edges of boxes , lower edges of boxes , upper whiskers and lower whiskers show median , third quartile , first quartile , maxima , and minima , respectively . P - values were calculated by the Student ' s t - test . ( * * * P - value < 0 . 001 ) .

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

(C) Quantification of brain lobe volume for the following genotypes: Lint-OKO -/+ (n = 42 biological replicates), Lint-OKO -/- (n = 36 biological replicates), and L(3)mbtts1 -/- (n = 34 biological replicates). Boxplot central bands, upper edges of boxes, lower edges of boxes, upper whiskers and lower whiskers show median, third quartile, first quartile, maxima, and minima, respectively. P-values were calculated by the Student's t-test. (***P-value < 0.001).


( D , E ) The genomic regions harboring the vasa ( D ) and aub ( E ) genes . The RNA - seq reads in y w , L ( 3 ) mbtts1 , and Lint - OKO brains are shown . The shading in gray corresponds to exons . The y - axis shows the number of RPM . RNA - seq samples were biological triplicates .

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

(D, E) The genomic regions harboring the vasa (D) and aub (E) genes. The RNA-seq reads in y w, L(3)mbtts1, and Lint-OKO brains are shown. The shading in gray corresponds to exons. The y-axis shows the number of RPM. RNA-seq samples were biological triplicates.


RT ‐ PCR performed on cDNA from RNA isolated from splenic B cells derived from Tam ‐ treated mb1 ‐ CreERT2 ( left lane ) or mb1 ‐ CreERT2 ; Sykfl / flmice ( right lane ) ; the Syk ‐ and Hprt ‐ specific amplification products were identified by agarose gel electrophoresis with Hprt used as an endogenous loading control .

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

RT‐PCR performed on cDNA from RNA isolated from splenic B cells derived from Tam‐treated mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/flmice (right lane); the Syk‐ and Hprt‐specific amplification products were identified by agarose gel electrophoresis with Hprt used as an endogenous loading control.


Immunoblot analysis of proteins isolated from B cells derived from spleens of mb1 ‐ CreERT2 ( left lane ) or mb1 ‐ CreERT2 ; Sykfl / fl ( right lane ) mice both treated with Tam as described ; blots were probed with anti ‐ Syk and anti ‐ GAPDH Ab , GAPDH being used as a loading control .

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

Immunoblot analysis of proteins isolated from B cells derived from spleens of mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/fl (right lane) mice both treated with Tam as described; blots were probed with anti‐Syk and anti‐GAPDH Ab, GAPDH being used as a loading control.


Intracellular flow cytometric analysis for Syk expression in B cells derived from the LN of Syk ‐ deficient and control mice .

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

Intracellular flow cytometric analysis for Syk expression in B cells derived from the LN of Syk‐deficient and control mice.


Genomic DNA analysis of Tam ‐ treated mb1 ‐ CreERT2 or mb1 ‐ CreERT2 ; Sykfl / flmice ( as indicated ) . Upper row : amplification of floxed ( fl ) and wt ( + ) alleles . Middle row : amplification of the deleted ( d ) allele . Lower row : loading control ( TC21 , a gene unaffected by the deletion of Syk ) . Established control DNA was used to visualize the individual bands ( as indicated ) .

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

Genomic DNA analysis of Tam‐treated mb1‐CreERT2 or mb1‐CreERT2;Sykfl/flmice (as indicated). Upper row: amplification of floxed (fl) and wt (+) alleles. Middle row: amplification of the deleted (d) allele. Lower row: loading control (TC21, a gene unaffected by the deletion of Syk). Established control DNA was used to visualize the individual bands (as indicated).


A , B Flow cytometric analysis of B cells from ( A ) the BM and ( B ) the SP of mb1 ‐ CreERT2 control ( left ) and mb1 ‐ CreERT2 ; Sykfl / flmice ( right ) treated with Tam as described in the Materials and Methods section . The BM cells were stained with anti ‐ IgM and anti ‐ B220 , and the SP cells with anti ‐ CD19 and anti ‐ CD93 or anti ‐ CD23 and anti ‐ CD21 . The gated regions in the dot blots correspond to individual B ‐ cell populations : ( A ) Bone marrow : gate P ( B220 + IgM − ) pro ‐ / pre ‐ B cells , gate I ( B220loIgM + ) immature B cells , gate M ( B220hiIgM + ) mature B cells ; ( B ) spleen : gate T ( CD19 + CD93 + ) transitional B cells ( T ) , gate M ( CD19 + CD93 − ) mature B cells ; in CD93 − splenic B cells , gate M ( CD23hiCD21int CD93 − ) mature follicular B cells and gate MZ ( CD23lo CD21hi CD93 − ) marginal zone B cells . The numbers in the dot plots indicate the mean relative frequency of cells in the gate . C - E Statistical analysis of absolute cell numbers per Tam ‐ treated mouse : filled circles indicating cells obtained from mb1 ‐ CreERT2 control mice and open circles from mb1 ‐ CreERT2 ; Sykfl / flmice . ( C ) Statistical analysis of the absolute cell numbers of pro ‐ / pre ‐ , immature and mature recirculating B cells in the BM . ( D ) Absolute cell numbers of transitional , M and MZ B cells found in the SP . ( E ) Statistical analysis of the absolute cells numbers of peritoneal B cell subsets B1 ‐ a and B1 ‐ b . An asterisk ( * ) marks statistically significant differences ( P 0 . 05 ) , two asterisks ( * * ) indicate P 0 . 01 , P ‐ values were obtained using two ‐ tailed Student ' s t ‐ test . Cell numbers of four to five mice per group are shown , with each dot representing an individual animal .

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

A, B Flow cytometric analysis of B cells from (A) the BM and (B) the SP of mb1‐CreERT2 control (left) and mb1‐CreERT2;Sykfl/flmice (right) treated with Tam as described in the Materials and Methods section. The BM cells were stained with anti‐IgM and anti‐B220, and the SP cells with anti‐CD19 and anti‐CD93 or anti‐CD23 and anti‐CD21. The gated regions in the dot blots correspond to individual B‐cell populations: (A) Bone marrow: gate P (B220+IgM−) pro‐/pre‐B cells, gate I (B220loIgM+) immature B cells, gate M (B220hiIgM+) mature B cells; (B) spleen: gate T (CD19+ CD93+) transitional B cells (T), gate M (CD19+ CD93−) mature B cells; in CD93−splenic B cells, gate M (CD23hiCD21int CD93−) mature follicular B cells and gate MZ (CD23lo CD21hi CD93−) marginal zone B cells. The numbers in the dot plots indicate the mean relative frequency of cells in the gate.C-E Statistical analysis of absolute cell numbers per Tam‐treated mouse: filled circles indicating cells obtained from mb1‐CreERT2 control mice and open circles from mb1‐CreERT2;Sykfl/flmice. (C) Statistical analysis of the absolute cell numbers of pro‐/pre‐, immature and mature recirculating B cells in the BM. (D) Absolute cell numbers of transitional, M and MZ B cells found in the SP. (E) Statistical analysis of the absolute cells numbers of peritoneal B cell subsets B1‐a and B1‐b. An asterisk (*) marks statistically significant differences (P 0.05), two asterisks (**) indicate P 0.01, P‐values were obtained using two‐tailed Student's t‐test. Cell numbers of four to five mice per group are shown, with each dot representing an individual animal.


A Flow cytometric analysis of IgM , CD19 and CD23 expression of splenic B cells from Tam ‐ treated mb1 ‐ CreERT2 control ( dashed line ) and mb1 ‐ CreERT2 ; Sykfl / flmice ( solid line ) .

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

A Flow cytometric analysis of IgM, CD19 and CD23 expression of splenic B cells from Tam‐treated mb1‐CreERT2 control (dashed line) and mb1‐CreERT2;Sykfl/flmice (solid line).


B Flow cytometric analysis of tyrosine phosphorylation ( pY ) in splenic B cells from Tam ‐ treated mb1 ‐ CreERT2 and mb1 ‐ CreERT2 ; Sykfl / flmice stimulated with medium ( dotted line ) or 50 μM pervanadate ( dashed and solid lines ) .

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

B Flow cytometric analysis of tyrosine phosphorylation (pY) in splenic B cells from Tam‐treated mb1‐CreERT2 and mb1‐CreERT2;Sykfl/flmice stimulated with medium (dotted line) or 50 μM pervanadate (dashed and solid lines).


C , D Flow cytometric analysis of the intracellular Ca2 + influx in purified splenic B cells derived from Tam ‐ treated mice . mb1 ‐ CreERT2 or mb1 ‐ CreERT2 ; Sykfl / fl were treated with ( C ) 10 μg / ml anti ‐ Kappa F ( ab ′ ) 2 fragments or ( D ) 1 μM ionomycin .

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

C, D Flow cytometric analysis of the intracellular Ca2+ influx in purified splenic B cells derived from Tam‐treated mice. mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl were treated with (C) 10 μg/ml anti‐Kappa F(ab′)2 fragments or (D) 1 μM ionomycin.


Ex vivo activation assay of splenic mature B cells from Tam ‐ treated mb1 ‐ CreERT2 ( upper row ) or Tam ‐ treated mb1 ‐ CreERT2 ; Sykfl / flmice ( lower row ) . Purified splenic B cells were either left unstimulated ( medium ) or were stimulated with the indicated stimuli . The cells were analyzed by flow cytometry after 24 h . Shown are CD19 versus CD86 dot plots . 7 ‐ AAD was included to distinguish dead from viable cells . The numbers in the quadrants indicate the relative frequencies of cells in the gate .

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

Ex vivo activation assay of splenic mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). Purified splenic B cells were either left unstimulated (medium) or were stimulated with the indicated stimuli. The cells were analyzed by flow cytometry after 24 h. Shown are CD19 versus CD86 dot plots. 7‐AAD was included to distinguish dead from viable cells. The numbers in the quadrants indicate the relative frequencies of cells in the gate.


Ex vivo proliferation assay with mature B cells from Tam ‐ treated mb1 ‐ CreERT2 ( upper row ) or Tam ‐ treated mb1 ‐ CreERT2 ; Sykfl / flmice ( lower row ) . The cells were incubated with the indicated stimuli for 90 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

Ex vivo proliferation assay with mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). The cells were incubated with the indicated stimuli for 90 h.


A Flow cytometric analysis of splenic B cells from Rag2 − / − ; γc − / − mice . CD19 versus CD3ε dot plots are shown . The mice were injected i . v . with 5 × 106 splenic ( CD19 + CD93 − ) B cells and ( CD3ε + ) T cells from Tam ‐ untreated mb1 ‐ CreERT2 control or mb1 ‐ CreERT2 ; Sykfl / fl mice . The recipient mice were treated with Tam beginning 1 day after transfer as described in the Materials and Methods section . B Quantitative analysis of B and T cells from Rag2 − / − ; γc − / − mice repopulated with mb1 ‐ CreERT2 or mb1 ‐ CreERT2 ; Sykfl / fl splenocytes . Each symbol represents an individual mouse . Filled circles and squares represent Syk + / + B and T cells , respectively ; open circles and squares represent Sykfl / fl B and T cells .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Flow cytometric analysis of splenic B cells from Rag2−/−;γc−/− mice. CD19 versus CD3ε dot plots are shown. The mice were injected i.v. with 5 × 106 splenic (CD19+ CD93−) B cells and (CD3ε+) T cells from Tam‐untreated mb1‐CreERT2 control or mb1‐CreERT2;Sykfl/fl mice. The recipient mice were treated with Tam beginning 1 day after transfer as described in the Materials and Methods section.B Quantitative analysis of B and T cells from Rag2−/−;γc−/− mice repopulated with mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl splenocytes. Each symbol represents an individual mouse. Filled circles and squares represent Syk+/+ B and T cells, respectively; open circles and squares represent Sykfl/fl B and T cells.


Demonstration of translational readthrough of AGO1 using luciferase - based reporter assay . Plasmid containing in - frame AGO1 - TGA ( or GCA ) - ISR - FLuc were transfected in HEK293 cells and translational readthrough was detected as FLuc activity normalized to the activity of co - transfected renilla luciferase ( RLuc ) . FLuc mRNA levels determined by RT - PCR are shown below . * * * , P = 0 . 0001 .

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

Demonstration of translational readthrough of AGO1 using luciferase-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-FLuc were transfected in HEK293 cells and translational readthrough was detected as FLuc activity normalized to the activity of co-transfected renilla luciferase (RLuc). FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0001.


Significance of the identity of the canonical stop codon in AGO1 translational readthrough . AGO1 - ISR - FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 cells and translational readthrough was quantified as described above . FLuc mRNA levels determined by RT - PCR are shown below . * * * , P = 0 . 0002 ; * * , P = 0 . 0004 .

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

Significance of the identity of the canonical stop codon in AGO1 translational readthrough. AGO1-ISR-FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 cells and translational readthrough was quantified as described above. FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0002; **, P = 0.0004.


First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough . AGO1 - TGA - FLuc constructs with different lengths of ISR ( all in - frame with AGO1 and FLuc ) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate . FLuc activity reflects readthrough activity . * * , P = 0 . 012 ; * , P = 0 . 017 .

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

First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough. AGO1-TGA-FLuc constructs with different lengths of ISR (all in-frame with AGO1 and FLuc) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate. FLuc activity reflects readthrough activity. **, P = 0.012; *, P = 0.017.


Demonstration of translational readthrough of AGO1 using fluorescence - based reporter assay . Plasmid containing in - frame AGO1 - TGA ( or GCA ) - ISR - GFP were transfected in HEK293 cells and translational readthrough was detected as fluorescence . Scale bar , 50 um . The bar graph shows mean fluorescence intensities in cells transfected with indicated constructs . Fluorescence intensity was measured by flowcytometry . * * * , P = 0 . 007 .

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

Demonstration of translational readthrough of AGO1 using fluorescence-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-GFP were transfected in HEK293 cells and translational readthrough was detected as fluorescence. Scale bar, 50 um. The bar graph shows mean fluorescence intensities in cells transfected with indicated constructs. Fluorescence intensity was measured by flowcytometry. ***, P = 0.007.


Detection of endogenous Ago1x by Western blot in HEK293 cells using anti - Ago1x antibody and pre - immune serum .

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

Detection of endogenous Ago1x by Western blot in HEK293 cells using anti-Ago1x antibody and pre-immune serum.


Western blot of lysates from HEK293 and HeLa cells using anti - Ago1x antibody pre - treated with Ago1x - specific peptide .

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

Western blot of lysates from HEK293 and HeLa cells using anti-Ago1x antibody pre-treated with Ago1x-specific peptide.


Expression of Ago1x in HEK293 cells transfected with three AGO1 - specific siRNAs ( 1 , 2 , 3 ) . qRT - PCR analysis of AGO1 transcript expression in these cells is shown below .

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

Expression of Ago1x in HEK293 cells transfected with three AGO1-specific siRNAs (1,2,3). qRT-PCR analysis of AGO1 transcript expression in these cells is shown below.


Proportion of Ago1x isoform in HEK293 cells . To separate Ago1x and Ago1 ( ~ 3 kDa difference ) , the cell lysate was electrophoresed for 3 . 5 h in 10 % polyacrylamide gel . Proportion of Ago1x and canonical isoform ( Ago1 ) was calculated by densitometric analysis . Results from three independent experiments are shown as a bar graph ( mean ± S . E . ) .

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

Proportion of Ago1x isoform in HEK293 cells. To separate Ago1x and Ago1 (~ 3 kDa difference), the cell lysate was electrophoresed for 3.5 h in 10% polyacrylamide gel. Proportion of Ago1x and canonical isoform (Ago1) was calculated by densitometric analysis. Results from three independent experiments are shown as a bar graph (mean ± S.E.).


Expression of Ago1x in mouse organs.


Presence of ribosome footprint in the Inter stop codon region of AGO1 transcript in U2 - OS cells . Ribosome profiling data was taken from NCBI ' s sequence read archive ( SRA Accession no . SRR1257257 ) and ribosome footprints on AGO1 transcript were analyzed as described in Methods section . Zoomed area near the inter - stop codon region is shown in the inset . Position of canonical stop codon and the downstream in - frame stop codon are shown . Detection of Ago1x by Western blot in U2 - OS cells is shown ( right ) .

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

Presence of ribosome footprint in the Inter stop codon region of AGO1 transcript in U2-OS cells. Ribosome profiling data was taken from NCBI's sequence read archive (SRA Accession no. SRR1257257) and ribosome footprints on AGO1 transcript were analyzed as described in Methods section. Zoomed area near the inter-stop codon region is shown in the inset. Position of canonical stop codon and the downstream in-frame stop codon are shown. Detection of Ago1x by Western blot in U2-OS cells is shown (right).


Effect of let - 7a inhibitor on translational readthrough of AGO1 . let - 7a inhibitor or control inhibitor was transfected in HeLa cells and the expression of Ago1x was determined by Western blot after 48 h . Densitometric analysis of three independent experiments is shown as a bar diagram ( mean ± S . E . ) . * , P = 0 . 01 .

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

Effect of let-7a inhibitor on translational readthrough of AGO1. let-7a inhibitor or control inhibitor was transfected in HeLa cells and the expression of Ago1x was determined by Western blot after 48 h. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.01.


Effect of let - 7a overexpression on translational readthrough of AGO1 . HeLa cells were stably transfected with pri - let - 7a overexpression construct . Ago1x expression was determined by Western blot . Densitometric analysis of three independent experiments is shown as a bar diagram ( mean ± S . E . ) . * , P = 0 . 007 .

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

Effect of let-7a overexpression on translational readthrough of AGO1. HeLa cells were stably transfected with pri-let-7a overexpression construct. Ago1x expression was determined by Western blot. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.007.


let - 7a inhibitor reduces the AGO1 readthrough efficiency . HeLa cells were transfected with let - 7a inhibitor or control inhibitor along with AGO1 - TGA ( or GCA ) - ISR - FLuc construct . Readthrough was quantified by measuring FLuc activity , which was normalized to co - transfected renilla luciferase activity . FLuc mRNA expression in HeLa cells treated with miRNA inhibitors is shown ( Right ) . * , P = 0 . 027 .

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

let-7a inhibitor reduces the AGO1 readthrough efficiency. HeLa cells were transfected with let-7a inhibitor or control inhibitor along with AGO1-TGA (or GCA)-ISR-FLuc construct. Readthrough was quantified by measuring FLuc activity, which was normalized to co-transfected renilla luciferase activity. FLuc mRNA expression in HeLa cells treated with miRNA inhibitors is shown (Right). *, P = 0.027.


AGO1 ISR with mutated let - 7a binding site shows reduced translational readthrough activity . Assay was done as described above in HeLa cells . Mutated sequence is shown ( top ) . * * * , P = 0 . 008 .

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

AGO1 ISR with mutated let-7a binding site shows reduced translational readthrough activity. Assay was done as described above in HeLa cells. Mutated sequence is shown (top). ***, P = 0.008.


AGO1 ISR with displaced let - 7a binding site ( AGO1 - TGA - disISR - FLuc ) shows reduced translational readthrough activity . let - 7a binding site was moved 18 nucleotides downstream of the stop codon . Readthrough assay was done as described above in HeLa cells . * * * , P < 0 . 0001 .

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

AGO1 ISR with displaced let-7a binding site (AGO1-TGA-disISR-FLuc) shows reduced translational readthrough activity. let-7a binding site was moved 18 nucleotides downstream of the stop codon. Readthrough assay was done as described above in HeLa cells. ***, P < 0.0001.


AGO1 ISR can drive translational readthrough in a heterologous context of RHOA . AGO1 coding sequence in AGO1 - TGA - ISR - FLuc construct was replaced by RHOA coding sequence ( 582 nucleotides ) and the readthrough assay was done as described above in HeLa cells . 3 ′ UTRRho , 159 nucleotides in the proximal 3 ′ UTR of RHOA ( NM _ 001664 ) ; ISRAgo1 , Inter - stop codon region of AGO1 . * * * , P < 0 . 0001 .

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

AGO1 ISR can drive translational readthrough in a heterologous context of RHOA. AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RHOA coding sequence (582 nucleotides) and the readthrough assay was done as described above in HeLa cells. 3′UTRRho, 159 nucleotides in the proximal 3′UTR of RHOA (NM_001664); ISRAgo1, Inter-stop codon region of AGO1. ***, P < 0.0001.


AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase ( RLuc ) . AGO1 coding sequence in AGO1 - TGA - ISR - FLuc construct was replaced by RLuc coding sequence and the readthrough assay was done as described above in HeLa cells ( left ) and in vitro using rabbit reticulocyte lysate system ( right ) . * * , P = 0 . 006 ; * * * , P = 0 . 0001 .

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

AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase (RLuc). AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RLuc coding sequence and the readthrough assay was done as described above in HeLa cells (left) and in vitro using rabbit reticulocyte lysate system (right). **, P = 0.006; ***, P = 0.0001.


Tethering of N - peptide - tagged Ago1 or Ago1x can induce readthrough of AGO1 . Two BoxB elements were cloned downstream of the AGO1 and upstream of the in - frame FLuc as shown in the schematic . This construct was transfected in HeLa cells expressing N - peptide - tagged or FLAG - tagged Ago proteins as indicated . Readthrough was measured as described above . * , P = 0 . 035 ( with Welch correction ) ; * * , P = 0 . 016 .

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

Tethering of N-peptide-tagged Ago1 or Ago1x can induce readthrough of AGO1. Two BoxB elements were cloned downstream of the AGO1 and upstream of the in-frame FLuc as shown in the schematic. This construct was transfected in HeLa cells expressing N-peptide-tagged or FLAG-tagged Ago proteins as indicated. Readthrough was measured as described above. *, P = 0.035 (with Welch correction); **, P = 0.016.


Linear regression analysis of miRNAs co - immunoprecipiated with Ago1 and those co - immunoprecipiated with Ago1x in HeLa cells . Reads per million ( RPM ) values of 395 miRNAs , which were common to Ago1 and Ago1x are shown .

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

Linear regression analysis of miRNAs co-immunoprecipiated with Ago1 and those co-immunoprecipiated with Ago1x in HeLa cells. Reads per million (RPM) values of 395 miRNAs, which were common to Ago1 and Ago1x are shown.


qRT - PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells . Bars show mean of three experiments ± S . E .

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

qRT-PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells. Bars show mean of three experiments ± S.E.


RT - PCR results showing PTEN and MYC mRNAs co - immunoprecipitated with Ago1 or Ago1x in HeLa cells .

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

RT-PCR results showing PTEN and MYC mRNAs co-immunoprecipitated with Ago1 or Ago1x in HeLa cells.


Result of co - immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells .

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

Result of co-immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells.


Result of co - immunoprecipitation experiment in HeLa cells shows that GW182 is co - immunoprecipitated with Ago1 , but not with Ago1x .

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

Result of co-immunoprecipitation experiment in HeLa cells shows that GW182 is co-immunoprecipitated with Ago1, but not with Ago1x.


Confocal immunofluoresence images of HeLa cells show co - localization of GW182 with Ago1 , but not with Ago1x . White circles indicate zoomed areas .

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

Confocal immunofluoresence images of HeLa cells show co-localization of GW182 with Ago1, but not with Ago1x. White circles indicate zoomed areas.


Confocal microscopy images of proximity ligation assay ( PLA ) in HeLa cells showing interaction of GW182 with Ago1 , but not with Ago1x . Rabbit anti - GW182 and Mouse anti - FLAG antibodies were used to achieve proximity ligation . Interaction is indicated by red dots that results from proximity ligation . Quantification of dots per cell is shown ( right ) . Each black circle represents a microscopic field . Mean ± S . E . are shown as horizontal lines ( n = 10 fields for vector control ; n = 12 fields for Ago1 ; n = 11 fields for Ago1x ) . At least 100 cells were counted for dots in each category . * * * , P < 0 . 0001 ( Mann - Whitney test ) .

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

Confocal microscopy images of proximity ligation assay (PLA) in HeLa cells showing interaction of GW182 with Ago1, but not with Ago1x. Rabbit anti-GW182 and Mouse anti-FLAG antibodies were used to achieve proximity ligation. Interaction is indicated by red dots that results from proximity ligation. Quantification of dots per cell is shown (right). Each black circle represents a microscopic field. Mean ± S.E. are shown as horizontal lines (n=10 fields for vector control; n=12 fields for Ago1; n=11 fields for Ago1x). At least 100 cells were counted for dots in each category. ***, P < 0.0001 (Mann-Whitney test).