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

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 presence of bafilomycin A1 ( BafA1 ) . ( C - E ) Transgenic C57BL / 6 mice expressing a GFP - LC3 fusion were injected with resveratrol spermidine at indicated concentrations . 3 h later , mice were killed , tissues were processed for immunofluorescence microscopy determinations of 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

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 percentages ( means ± ; n = 3 ; * , P < 0 . 05 ; * * , P < 0 . 01 as compared with same tissue from untreated ) of 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

(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 p62 content in WT C57BL / 6 mouse tissues . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

O O O 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) 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 changing PD - dependently in Columbia LCC cohorts . Only quantified in both cohorts are shown ( n = 330 ) . overlapping between 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

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 33 that were LRRK2 - dependently regulated in both cohorts using a pairwise t - comparing four subgroups ( HC , NMC , iPD LRRK2 PD ) .

O O O O O O O O O O O O O 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) 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 associated p - values [ - log10 ] of intensities with MoCA total score . Either PD ( left ) , iPD ( middle ) or LRRK2 PD ( right ) were included in . Significantly correlated 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 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 associated p - values [ - log10 ] of intensities with UPDRS - III score . Either PD ( left ) , iPD ( middle ) or LRRK2 PD ( right ) were included . Significantly correlated 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.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by - activated sorting for with high low autophagic flux using of mCherry / GFP ( a ) . high low 20 % were sorted ( a ) , re - plated treated with lysosomal protease inhibitors pepstatin E - 64d for 1 h ; lysates were then immunoblotted for indicated ( b ) . ( c ) Densitometry of LC3 - II p62 western blots ( normalized to actin 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were assessed by quantitative

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of HeLa mCherry - GFP - LC3 sorted for autophagic flux as in a . Yellow arrows denote autophagosomes ; red arrows indicate autolysosomes . ( g ) of autophagosomes autolysomes from electron ( mean ± s . e . m . , n = 50 fields ) . Uncropped 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

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 stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by - activated sorting for with high low autophagic flux using of mCherry / GFP . were then re - plated in growth medium autophagic flux was again measured by flow cytometry at indicated time points ( median of mCherry / GFP ± s . e . m . , n = 3 wells ) . Lysates from collected at indicated time points were immunoblotted with indicated antibodies ( b ) . Representative flow cytometry histograms for at 0 h 24 h after sorting ( 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 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 were sorted for autophagic flux as in a ( top bottom 20 % ) . Following treatment with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) , viability was determined by MTS ( a tetrazolium reduction - metabolic measurement ) at 24 h ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 sorted for autophagic flux followed by treatment with Fas ligand ( 4 ng ml − 1 ) or TRAIL ( 15 ng ml − 1 ) for 24 h . were then re - plated allowed to recover for 5 ( Fas ligand ) or 6 ( TRAIL ) days then assayed for viability ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were sorted for autophagic flux as above apoptosis was measured at 1 h by flow cytometry using AnnexinV DAPI ( mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) sorted as in a , were re - plated , starting at indicated times following sorting , treated with Fas ligand ( 4 ng ml − 1 ) for 24 h ; viability was then determined by MTS ( of untreated control , mean ± s . e . m . , n = 3 wells ) . Uncropped 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

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

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

(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 in a , c probed for indicated antibodies . CQ ,

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


( c ) BJAB Jurkat 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 ) . were then re - plated at low density in growth media allowed to recover for 5 days , then assayed for viability ( of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0024 ) .

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

(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 Jurkat were transduced with indicated lentiviral shRNA constructs , followed by 3 days of puromycin selection . Selected were plated treated with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) for 24 h viability was assessed by MTS ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 p62 confirm depletion autophagy inhibition in d , f .

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


( f ) BJAB Jurkat 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 ) . were then re - plated at low density allowed to recover for 5 - 6 days , then assayed for viability ( 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 growth suppressive effect of Atg7 knockdown in absence of drug treatment . Uncropped 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Jurkat stably expressing mCherry - GFP - LC3 were flow sorted for high low autophagic flux , treated with Fas ligand ( 1 . 5 ng ml − 1 ) for 24 h viability was assessed by MTS ( a ; of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) . ( b ) Immunoblots following sorting of samples in a .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) indicated 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 collected after chloroquine treatment were immunoblotted with indicated antibodies ( c ) .

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 B-CONTROLLED_VAR 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 ) indicated 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 collected after chloroquine treatment were immunoblotted with indicated antibodies ( c ) . viability was assessed by MTS 24 h following Fas ligand treatment ( d ; 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 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 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

(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 Jurkat were transduced with control , Atg5 , Atg7 or Vps34 shRNA lentiviruses , followed by three days of puromycin selection . were then treated with Fas ligand ( 1 . 25 ng ml − 1 ) or TRAIL ( 1 . 25 ng ml − 1 ) for 24 h viability was assessed by MTS ( e ; 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 Vps34 knockdown , autophagy inhibition altered Fap - 1 . blots are separated because different exposures were required to detect in one without overexposing lanes for other ; Fap - 1 blot is not separated to demonstrate lack of Fap - 1 in Type II Jurkat ( f ) . Fap - 1 blots ( b , c , f ) were run on separate gels owing to quantity of required for detection ( see Methods ) . Uncropped 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Jurkat expressing indicated Fap - 1 wild - type ( WT ) 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 . viability was determined by MTS ( a ; of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 2 . 4 × 10 − 5 , * * * P = 0 . 0024 ) . lysates were blotted probed with indicated antibodies ( b ) .

O O O 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

(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 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 ; viability was then determined by MTS ( c ; 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 ) .

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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 from c by re - plating them at low density allowing them to recover for 5 days , followed by viability ( 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 quantity of required for detection ( see Methods ) . Uncropped of blots are shown in Supplementary Fig . 6 .

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(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 ) viability was determined by MTS following Fas ligand ( 4 ng ml − 1 ) treatment in BJAB expressing control or p62 lentiviral shRNAs ( a ; 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 Fap - 1 ( b ) .

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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 Fap - 1 in BJAB treated with 20 μM chloroquine for 16 h , followed by treatment with Fas ligand ( 50 ng ml − 1 ) for 2 h at 4 ° C .

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

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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 HDAC6 KO MEFs were treated with MG132 subjected to Western blot for LC3 , actin , HDAC6 .

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


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

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

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(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 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 red signals indicate acidic autophagolysosomes . Scale bar , 10 μm .

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

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

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

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(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 ) of autophagosomes autophagolysosomes . error bar represents standard error .

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


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

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

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

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

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

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

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

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

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(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 . Different subcellular fractions ( 75 μg ) isolated from wild type ( WT ) were subjected to SDS - PAGE immunoblotting for indicated . Hom , homogenate ; APG , autophagosomes ; APL , autophagolysosomes ; Lys , lysosomes .

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

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

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

(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 , stained with antibodies for Lap ‐ 1 ( red , to label lysosome ) , or ubiquitin ( green ) phalloidin for actin ( blue ) . Note that F ‐ actin at was lost , but lysosomes remained concentrated in cortactin knockdown ( arrow ) .

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(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 , subjected to filter ‐ trap using a ubiquitin antibody . knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody against cortactin GAPDH in right panel .

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

(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 ) 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 .

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(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 in brain . hippocampus cerebral cortex regions from 6 ‐ month ‐ old wild ‐ type ubiquitin KO were subjected to immunostaining with a ubiquitin antibody counterstained with hematoxylin . red arrows indicate ubiquitin ‐ positive neuritic black arrows indicate . These ubiquitin ‐ positive structures were rarely observed in control . Scale bar , 50

(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 in cortex hippocampus region of HDAC6 KO mice as determined by TUNEL . Apoptotic were only observed in HDAC6 KO mice . Scale bar , 100 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.


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

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

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

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

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

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(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 OSC lysates . n . i . : non - immune IgG .

O O O O O O O O O O O O O O O O O O O O O O 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) 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 its ∆ SAM mutant ( green ) . Both were localized to nuclei ( DAPI : blue ) . Scale bar : 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 OSC lysates . n . i . : non - immune IgG .

O O O O O O O O O O O O O O O O O O O O O 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) 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 its ∆ SAM mutant ( green ) . Both were localized to nuclei ( DAPI : blue ) . Scale bar : 5 μm .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 its 8CA mutant , but not with ∆ PHD mutant An asterisk shows background .

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

(H) 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 its 8CA ∆ PHD mutants ( green ) . were localized to nuclei ( DAPI : blue ) . Scale bar : 5 μm .

(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 vasa promoter was weakened after loss of Lint - O . Data represent means ± SE ( n = 3 biological replicates ) . p values were calculated with t - . * * p : < 0 . 01 . t - were performed against samples with ⎾ symbol .

(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 amounts of L ( 3 ) mbt , Lint - O , histone H3 ( H3 ) in normal ( control ) , L ( 3 ) mbt - depleted ( RNAi ) , Lint - O - depleted ( RNAi ) OSCs .

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

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

(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 shows mRNA of lint - O in y w Lint - OKO ovaries . Data are expressed as mean error bars represent SD . n = 2 biological replicates .

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

(B) RT-qPCR analysis 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 of y w Lint - OKO ovaries immunostained for Vasa . Scale bar : 50 μm .

O O O O O O O O O O O O O O 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 of y w Lint - OKO ovarioles immunostained for Vasa . Vasa was ectopically expressed in follicle ( arrowheads ) . Scale bar : 50 μm .

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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 isolated from splenic B derived from Tam ‐ treated mb1 ‐ CreERT2 ( left lane ) or mb1 ‐ CreERT2 ; Sykfl / flmice ( right lane ) ; Syk ‐ 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 O O O O O O 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 of isolated from B 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 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 O O O O O O O O O O O O O O O O O O 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 for Syk expression in B derived from LN of Syk ‐ deficient control mice .

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


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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of IgM , CD19 CD23 expression of splenic B from Tam ‐ treated mb1 ‐ CreERT2 control ( dashed ) mb1 ‐ CreERT2 ; Sykfl / flmice ( solid ) .

O O O O O O O O O O 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 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 of tyrosine phosphorylation ( pY ) in splenic B from Tam ‐ treated mb1 ‐ CreERT2 mb1 ‐ CreERT2 ; Sykfl / flmice stimulated with medium ( dotted ) or 50 μM pervanadate ( dashed 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 O O O O O O O O O B-CONTROLLED_VAR 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 of intracellular Ca2 + influx in purified splenic B 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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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

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 of splenic mature B from Tam ‐ treated mb1 ‐ CreERT2 ( upper row ) or Tam ‐ treated mb1 ‐ CreERT2 ; Sykfl / flmice ( lower row ) . Purified splenic B were either left unstimulated ( medium ) or were stimulated with indicated stimuli . were analyzed by flow cytometry after 24 h . Shown are CD19 versus CD86 dot plots . 7 ‐ AAD was included to distinguish dead from viable . numbers in quadrants indicate relative frequencies of in 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 with mature B from Tam ‐ treated mb1 ‐ CreERT2 ( upper row ) or Tam ‐ treated mb1 ‐ CreERT2 ; Sykfl / flmice ( lower row ) . were incubated with 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 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 of splenic B from Rag2 − / − ; γc − / − mice . CD19 versus CD3ε dot plots are shown . mice were injected i . v . with 5 × 106 splenic ( CD19 + CD93 − ) B ( CD3ε + ) T from Tam ‐ untreated mb1 ‐ CreERT2 control or mb1 ‐ CreERT2 ; Sykfl / fl mice . recipient mice were treated with Tam beginning 1 day after transfer as described in Materials Methods section . B Quantitative of B T from Rag2 − / − ; γc − / − mice repopulated with mb1 ‐ CreERT2 or mb1 ‐ CreERT2 ; Sykfl / fl splenocytes . Each symbol represents an mouse . Filled circles squares represent Syk + / + B T , respectively ; open circles squares represent Sykfl / fl B T .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ( in - frame with AGO1 FLuc ) were in vitro transcribed 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - reporter . Plasmid containing in - frame AGO1 - TGA ( or GCA ) - ISR - GFP were transfected in HEK293 translational readthrough was detected as . Scale bar , 50 um . bar graph shows mean intensities in transfected with indicated constructs . was measured by flowcytometry . * * * , P = 0 . 007 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 using anti - Ago1x antibody pre - immune serum .

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


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

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

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

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

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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 expression of Ago1x was determined by Western blot after 48 h . Densitometric of three independent experiments is shown as a bar diagram ( mean ± S . E . ) . * , P = 0 . 01 .

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

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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 AGO1 readthrough efficiency . HeLa 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 treated with miRNA inhibitors is shown ( Right ) . * , P = 0 . 027 .

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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 . was done as described above in HeLa . Mutated is shown ( top ) . * * * , P = 0 . 008 .

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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 stop codon . Readthrough was done as described above in HeLa . * * * , P < 0 . 0001 .

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

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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 in AGO1 - TGA - ISR - FLuc construct was replaced by RLuc coding readthrough was done as described above in HeLa ( left ) in vitro using rabbit reticulocyte lysate system ( right ) . * * , P = 0 . 006 ; * * * , P = 0 . 0001 .

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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 AGO1 upstream of in - frame FLuc as shown in schematic . This construct was transfected in HeLa expressing N - peptide - tagged or FLAG - tagged Ago as indicated . Readthrough was measured as described above . * , P = 0 . 035 ( with Welch correction ) ; * * , P = 0 . 016 .

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

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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 enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa . Bars show mean of three experiments ± S . E .

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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 MYC mRNAs co - immunoprecipitated with Ago1 or Ago1x in HeLa .

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


Result of co - immunoprecipitation experiments demonstrating interaction of Dicer with Ago1 Ago1x in HeLa .

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 shows that GW182 is co - immunoprecipitated with Ago1 , but not with Ago1x .

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


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

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


Confocal microscopy of proximity ligation ( PLA ) in HeLa showing interaction of GW182 with Ago1 , but not with Ago1x . Rabbit anti - GW182 Mouse anti - FLAG antibodies were used to achieve proximity ligation . Interaction is indicated by red dots that results from proximity ligation . of dots per 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 were counted for dots in each category . * * * , P < 0 . 0001 ( Mann - Whitney ) .

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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).


Activity of RLuc relative to co - transfected FLuc in HeLa expressing N - HA - tagged or untagged Ago as indicated . Bar graph ( mean ± S . E . ) is a representative of four independent experiments done in triplicates . Expression of HA - tagged Ago was confirmed by Western blot ( bottom ) . * , P = 0 . 018 ; # , P = 0 . 041 ; $ , P = 0 . 013 . Numbers on bars represent % change in relative luciferase activity .

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Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Bar graph (mean ± S.E.) is a representative of four independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). *, P = 0.018; #, P = 0.041; $, P = 0.013. Numbers on the bars represent % change in relative luciferase activity.


Expression of RLuc in HeLa expressing N - HA - tagged or untagged Ago as indicated . Densitometric of three independent experiments is shown as bar graph ( mean ± S . E . ) . RT - PCR for RLuc mRNA level is shown ( bottom ) . * * , P = 0 . 001 .

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Expression of RLuc protein in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). RT-PCR analysis for RLuc mRNA level is shown (bottom). **, P = 0.001.


Activity of RLuc relative to co - transfected FLuc in HeLa expressing N - HA - tagged Ago2 ( N - HA - Ago2 ) or N - HA - tagged chimeric Ago2 appended with ISR region of Ago1x ( N - HA - Ago2 - ISRAgo1 ) as indicated . Bar graph ( mean ± S . E . ) is a representative of three independent experiments done in triplicates . Expression of HA - tagged Ago was confirmed by Western blot ( bottom ) . * * * , P < 0 . 0001 ; * * , P = 0 . 0004 .

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Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged Ago2 (N-HA-Ago2) or N-HA-tagged chimeric Ago2 appended with the ISR region of Ago1x (N-HA-Ago2-ISRAgo1) as indicated. Bar graph (mean ± S.E.) is a representative of three independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). ***, P < 0.0001; **, P = 0.0004.


Ribopuromycylation showing global translation profile of HeLa overexpressing FLAG - HA - Ago1 or FLAG - HA - Ago1x . Densitometric of four independent experiments is shown as bar graph ( mean ± S . E . ) . Puromycin densities were normalized to ponceau S stain densities . * , P < 0 . 029 ( Mann - Whitney ) .

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Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of four independent experiments is shown as bar graph (mean ± S.E.). Puromycin densities were normalized to ponceau S stain densities. *, P < 0.029 (Mann-Whitney test).


[ 35S ] - methionine autoradiogram showing global translation profile of HeLa overexpressing FLAG - HA - Ago1 or FLAG - HA - Ago1x . Densitometric of three independent experiments is shown as bar graph ( mean ± S . E . ) . Autoradiogram densities were normalized to Coomassie stain densities . * , P < 0 . 029 ( Mann - Whitney ) .

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[35S]-methionine autoradiogram showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). Autoradiogram densities were normalized to Coomassie stain densities. *, P < 0.029 (Mann-Whitney test).


A Pancreatic sections ( 20X magnification ) from 10 - months old WT Glo1KD mice stained with hematoxylin eosin ( H & E ) , anti - insulin , anti - glucagon anti - F4 / 80 antibodies , are representative of sections from 7 mice each group .

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A Pancreatic sections (20X magnification) from 10-months old WT and Glo1KD mice stained with hematoxylin and eosin (H&E), anti-insulin, anti-glucagon and anti-F4/80 antibodies, are representative of sections from 7 mice each group.


H , I J Insulin glucagon IS , calculated as score multiplied with of positive ( H , I ) of islets stained with F4 / 80 ( J ) ( n = 7 mice for each genotype ) . Data information : box plots show minimum maximum values ( ends of whiskers ) , interquartile range ( of box ) , median ( through box ) mean ( + ) values . * p ≤ 0 . 05 , * * p ≤ 0 . 01 ( Mann - Whitney ) .

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H, I and J Insulin and glucagon IS, calculated as the intensity score multiplied with percentage of positive cells (H, I) and the percentage of islets area stained with F4/80 (J) (n=7 mice for each genotype). Data information: box plots show the minimum and maximum values (ends of the whiskers), interquartile range (length of the box), median (line through the box) and mean (+) values. *p≤0.05, **p≤0.01 (Mann-Whitney test).


C Whole lysates of INS - 1 832 / 13 β - analysed by Western Blot with anti - p53 anti - p21 antibodies ; anti - vinculin antibody was used as loading control ( n = 9 biological replicates for each treatment group ) . Data information : Data in bar graphs are presented as mean ± . In C D , blots show representative image of replicate experiments ; were quantified by densitometric of independent experiments showed in bar graphs as over not treated control . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 : MGO vs CTR ; # p ≤ 0 . 05 , # # p ≤ 0 . 01 , # # # p ≤ 0 . 001 : D / Q vs MGO ( Mann - Whitney

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C Whole cell protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-p53 and anti-p21 antibodies; anti-vinculin antibody was used as loading control (n=9 biological replicates for each treatment group). Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO (Mann-Whitney test


D lysates of INS - 1 832 / 13 β - analysed by Western Blot with anti - HMGB1 antibody ; anti - histone H3 antibody was used as loading control ( n = 5 biological replicates for each treatment group ) . Data information : Data in bar graphs are presented as mean ± . In C D , blots show representative image of replicate experiments ; were quantified by densitometric of independent experiments showed in bar graphs as over not treated control . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 : MGO vs CTR ; # p ≤ 0 . 05 , # # p ≤ 0 . 01 , # # # p ≤ 0 . 001 : D / Q vs MGO ( Mann - Whitney

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D Nuclear protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-HMGB1 antibody; anti-histone H3 antibody was used as loading control (n=5 biological replicates for each treatment group). Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO (Mann-Whitney test


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . of IMM in living HeLa , L6 , H1975 , stained with 10 - N - nonyl acridine orange ( NAO ) . NAO preferentially binds phospholipids in IMM , such as cardiolipin . Arrowheads indicate cristae in IMM . Scale bar = 500 nm . N ≥ 3 independent experiments for each type .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Images of IMM in living HeLa, L6, and H1975 cells, stained with 10-N-nonyl acridine orange (NAO). NAO preferentially binds phospholipids in the IMM, such as cardiolipin. Arrowheads indicate cristae in the IMM. Scale bar = 500 nm. N ≥ 3 independent experiments for each cell type.


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . Mitochondrion cropped from HeLa shown in A ( dashed ) zoomed in to show cristae , inner boundary ( IBM ) , matrix ( arrows ) . Scale bar = 100 nm .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondrion cropped from HeLa cell shown in A (dashed line) and zoomed in to show cristae, inner boundary membrane (IBM), and matrix (arrows). Scale bar = 100 nm.


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . H1975 transduced with matrix - targeted - DsRed stained with NAO . Matrix - targeted Ds - Red differentiates matrix from cristae stained with NAO . Arrowheads point to cristae . Scale bar = 500 nm . N = 1 independent experiment .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. H1975 cells transduced with matrix-targeted-DsRed and stained with NAO. Matrix-targeted Ds-Red differentiates matrix from cristae stained with NAO. Arrowheads point to cristae. Scale bar = 500 nm. N = 1 independent experiment.


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . structure of IMM in Mic13 - KO ( HeLa ) , stained with NAO . number of cristae is decreased in Mic13 - KO compared to control shown in panel A , labeled with arrowheads . Scale bar = 500 nm . N = 3 independent experiments .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. The structure of the IMM in Mic13-KO cells (HeLa), stained with NAO. The number of cristae is decreased in Mic13-KO compared to control cells shown in panel A, labeled with arrowheads. Scale bar = 500 nm. N = 3 independent experiments.