Figure 3Representative images of rat neonatal CMs 4 days following transfection with a FLAG - tagged TRF1 - FokI - D450A ( top row ) or TRF1 - FokI ( middle and bottom row ) fusion protein ( cell treatments the same for all subsequent panels in Figure ) ( red - telo - FISH ; green - γH2A . X ) . Images are z - projections of 0 . 1µm stacks taken with 100X objective . White arrows indicate co - localisation between telomeres and γH2A . X , with co - localising foci amplified in the right panels ( taken from single z - planes where co - localisation was found ) . Scale bar represents 3 . 5μm . Scale bar in magnified images showing individual co - localisation 500nm . % of γH2A . X foci co - localising with telomeres ( B ) in FLAG - tagged TRF1 - FokI - D450A - and TRF1 - FokI - expressing CMs . Data are mean ± SEM of n = 4 independent experiments . > 50 cells were analysed per condition . Statistical analysis was performed by two - tailed t - test * * * P < 0 . 001 . mean number of Telomere - associated Foci ( TAF ) ( C ) in FLAG - tagged TRF1 - FokI - D450A - and TRF1 - FokI - expressing CMs . Data are mean ± SEM of n = 4 independent experiments . > 50 cells were analysed per condition . Statistical analysis was performed by two - tailed t - test * * * P < 0 . 001 . Histograms displaying telomere intensity for telomeres co - localising or not co - localising with γH2AX foci . Red dotted lines represent median . Mann - Whitney test show no significant difference in telomere intensity between TAF and non - TAF . Mean % of FLAG - labelled CMs positive for SA - β - Gal activity . Data are mean ± SEM of n = 3 independent experiments . > 100 cells were quantified per condition . Statistical analysis performed using two tailed t test ; * * P < 0 . 01 . Expression of p21 mRNA ( as a function of β - actin and Gapdh ) by real - time PCR in TRF1 - FokI - D450A and TRF1 - FokI expressing CMs . Data are mean ± SEM of n = 6 independent experiments . Statistical analysis performed using two tailed t test ; * * * P < 0 . 001 . Mean ± SEM cell surface area ( μm2 ) of FLAG - labelled CMs expressing TRF1 - FokI - D450A and TRF1 - FokI . Statistical analysis performed using two tailed t test ; * * * P < 0 . 001 . Scale bar represents 50μm .

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Figure 3Representative images of rat neonatal CMs 4 days following transfection with a FLAG-tagged TRF1-FokI-D450A (top row) or TRF1-FokI (middle and bottom row) fusion protein (cell treatments the same for all subsequent panels in Figure) (red - telo-FISH; green - γH2A.X). Images are z-projections of 0.1µm stacks taken with 100X objective. White arrows indicate co-localisation between telomeres and γH2A.X, with co-localising foci amplified in the right panels (taken from single z-planes where co-localisation was found). Scale bar represents 3.5μm. Scale bar in magnified images showing individual co-localisation 500nm.% of γH2A.X foci co-localising with telomeres (B) in FLAG-tagged TRF1-FokI-D450A- and TRF1-FokI-expressing CMs. Data are mean ± SEM of n=4 independent experiments. >50 cells were analysed per condition. Statistical analysis was performed by two-tailed t-test ***P<0.001.mean number of Telomere-associated Foci (TAF) (C) in FLAG-tagged TRF1-FokI-D450A- and TRF1-FokI-expressing CMs. Data are mean ± SEM of n=4 independent experiments. >50 cells were analysed per condition. Statistical analysis was performed by two-tailed t-test ***P<0.001.Histograms displaying telomere intensity for telomeres co-localising or not co-localising with γH2AX foci. Red dotted lines represent median. Mann-Whitney test show no significant difference in telomere intensity between TAF and non-TAF.Mean % of FLAG-labelled CMs positive for SA-β-Gal activity. Data are mean ± SEM of n=3 independent experiments. >100 cells were quantified per condition. Statistical analysis performed using two tailed t test; **P<0.01.Expression of p21 mRNA (as a function of β-actin and Gapdh) by real-time PCR in TRF1-FokI-D450A and TRF1-FokI expressing CMs. Data are mean ± SEM of n=6 independent experiments. Statistical analysis performed using two tailed t test; ***P<0.001.Mean± SEM cell surface area (μm2) of FLAG-labelled CMs expressing TRF1-FokI-D450A and TRF1-FokI. Statistical analysis performed using two tailed t test; ***P<0.001. Scale bar represents 50μm.


Figure 4Real - time PCR gene expression analysis in isolated mouse CMs from C57BL / 6 mice . Mean % of p21 - positive CM nuclei from 3 , 15 , and 30 month old C57BL / 6 mice by immunohistochemistry ( IHC ) . Data are mean ± SEM of n = 4 per age group . > 100 CMs were quantified per age group . Mean % of 3 and 24 month old mouse CMs staining positive for SA - β - Gal in vivo with representative images above ( blue - SA - β - Gal ; green - troponin C ; red - WGA ) . Black arrows indicate SA - β - Gal expression in a troponin C expressing CM . Statistical analysis performed using two - tailed t - test ; * P < 0 . 05 . Data are mean ± SEM from the analysis of > 500 CMs per mouse , 4 mice per age group . Scale bar 20µm . Mean % of SADS - positive CM nuclei from 3 and 30 month old mouse CMs positive for SADS in vivo , as detected by centromere - FISH . Data are mean ± SEM of n = 4 per age group . > 200 CMs per mouse were quantified . SASP heatmap : Pearson correlation clustered heatmap showing a curated list of known SASP genes ( top panel ) or a selection of secreted SASP proteins ( bottom panel ) in young ( 3 months ) and old ( 20 months ) mouse CMs ( n = 5 per age group ) . The colour intensity represents column Z - score , where red indicates highly and blue lowly expressed .

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Figure 4Real-time PCR gene expression analysis in isolated mouse CMs from C57BL/6 mice.Mean % of p21-positive CM nuclei from 3, 15, and 30 month old C57BL/6 mice by immunohistochemistry (IHC). Data are mean ± SEM of n=4 per age group. >100 CMs were quantified per age group.Mean % of 3 and 24 month old mouse CMs staining positive for SA-β-Gal in vivo with representative images above (blue - SA-β-Gal; green - troponin C; red - WGA). Black arrows indicate SA-β-Gal expression in a troponin C expressing CM. Statistical analysis performed using two-tailed t-test; *P<0.05. Data are mean ± SEM from the analysis of >500 CMs per mouse, 4 mice per age group. Scale bar 20µm.Mean % of SADS-positive CM nuclei from 3 and 30 month old mouse CMs positive for SADS in vivo, as detected by centromere-FISH. Data are mean ± SEM of n=4 per age group. >200 CMs per mouse were quantified.SASP heatmap: Pearson correlation clustered heatmap showing a curated list of known SASP genes (top panel) or a selection of secreted SASP proteins (bottom panel) in young (3 months) and old (20 months) mouse CMs (n=5 per age group). The colour intensity represents column Z-score, where red indicates highly and blue lowly expressed.


Figure 5 ( Above ) RNA - sequencing of purified CMs from 4 mice per age group reveal age - dependent increased expression of 3 secreted proteins Edn3 , Tgfb2 , and Gdf15 ; the colour intensity represents column Z - score , where red indicates highly and blue lowly expressed . ( Below ) mRNA expression of Edn3 , Tgfb2 , and Gdf15 was independently validated by RT - PCR in young and old isolated adult CMs . Data are mean ± S . E . M of n = 8 mice per group . Representative images of immunofluorescent staining against α - SMA and EdU in neonatal fibroblasts cultured in the presence of CM from young and old CMs . Quantification of % of EdU incorporation ( D ) in neonatal fibroblasts after treatment for 48h with CM from young and old CM . Data are mean ± S . E . M of n = 3 - 4 mice per age group ; > 200 cells were quantified per condition . % of a - SMA positive cells ( E ) in neonatal fibroblasts after treatment for 48h with CM from young and old CM . Data are mean ± S . E . M of n = 3 - 4 mice per age group ; > 200 cells were quantified per condition . Representative micrographs of neonatal fibroblasts and CMs treated with recombinant proteins : Tgfb2 , Edn3 , and Gdf15 for 48h and immunostained against α - SMA and EdU ( fibroblasts ) and α - actinin ( CMs ) . Quantification of α - SMA ( G ) in neonatal CMs following treatment with the indicated recombinant proteins . Data are mean ± S . E . M of n = 3 - 4 independent experiments . EdU positive neonatal fibroblasts ( H ) in neonatal CMs following treatment with the indicated recombinant proteins . Data are mean ± S . E . M of n = 3 - 4 independent experiments . surface area ( μm2 ) ( I ) in neonatal CMs following treatment with the indicated recombinant proteins . Data are mean ± S . E . M of n = 3 - 4 independent experiments .

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Figure 5(Above) RNA-sequencing of purified CMs from 4 mice per age group reveal age-dependent increased expression of 3 secreted proteins Edn3, Tgfb2, and Gdf15; the colour intensity represents column Z-score, where red indicates highly and blue lowly expressed. (Below) mRNA expression of Edn3, Tgfb2, and Gdf15 was independently validated by RT-PCR in young and old isolated adult CMs. Data are mean ±S.E.M of n=8 mice per group.Representative images of immunofluorescent staining against α-SMA and EdU in neonatal fibroblasts cultured in the presence of CM from young and old CMs.Quantification of % of EdU incorporation (D) in neonatal fibroblasts after treatment for 48h with CM from young and old CM. Data are mean ±S.E.M of n=3-4 mice per age group; >200 cells were quantified per condition.% of a-SMA positive cells (E) in neonatal fibroblasts after treatment for 48h with CM from young and old CM. Data are mean ±S.E.M of n=3-4 mice per age group; >200 cells were quantified per condition.Representative micrographs of neonatal fibroblasts and CMs treated with recombinant proteins: Tgfb2, Edn3, and Gdf15 for 48h and immunostained against α-SMA and EdU (fibroblasts) and α-actinin (CMs).Quantification of α-SMA (G) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments.EdU positive neonatal fibroblasts (H) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments.surface area (μm2) (I) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments.


Figure 6Mito and ETC genes with GSEA analysis : Clustered heatmap showing all genes associated with the " Mitochondrion " GO term in young and old , mouse CMs as observed by the GSEA pre - ranked list enrichment analysis ( normalised enrichment score : - 1 . 70 ; FDR q - value < 0 . 05 ) . Alongside this is a column clustered heatmap displaying a list of genes from the electron transport chain ( ETC ) GO ontology . In both instances , genes are by column and samples by row with the colour intensity representing column Z - score , where red indicates highly and blue lowly expressed . Real - time PCR gene expression analysis of MAO - A , MnSOD and catalase in isolated mouse CMs from young ( 3 months ) and old ( 20 months ) mice . Mean % of 4 - HNE - ( top ) or 8oxodG - ( bottom ) positive CMs from 3 month ( young ) or 30 month ( old ) aged mice . Data are mean ± SEM of n = 4 per age group . 100 CMs were quantified per age group . Mean % of TAF - positive nuclei ( left graphs ) or mean % of TAF ( right graphs ) in wild - type ( control ) compared to MAO - A transgenic mice with or without drinking water supplemented with 1 . 5g / kg / day NAC from the age of 4 to 24 weeks Data are mean ± S . E . M of n = 3 - 4 per group . > 100 CMs were quantified per age group . Mean % of TAF - positive nuclei ( left graphs ) or mean % of TAF ( right graphs ) in MnSOD + / + vs MnSOD - / + , Data are mean ± S . E . M of n = 3 - 4 per group . > 100 CMs were quantified per age group . Mean % of TAF - positive nuclei ( left graphs ) or mean % of TAF ( right graphs ) in Catalase + / + vs . Catalase - / - , Data are mean ± S . E . M of n = 3 - 4 per group . > 100 CMs were quantified per age group . Mean % of TAF - positive nuclei ( left graphs ) or mean % of TAF ( right graphs ) in WT vs . POLG double mutant mice . Data are mean ± S . E . M of n = 3 - 4 per group . > 100 CMs were quantified per age group . Schematic depicting isolated mouse adult CMs isolated from 4 animals were treated with or without 100nM rotenone either in the presence of 5mM NAC or vehicle control ( pre - treated for 30 minutes before rotenone treatment ) , for 24 h before fixation . Mean number of TAF ( top graph ) and mean % of TAF - positive nuclei ( bottom graph ) . Data are mean ± SEM from 4 separate CM cultures isolated from 3 month - old mice . 50 CMs were quantified per condition .

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Figure 6Mito and ETC genes with GSEA analysis: Clustered heatmap showing all genes associated with the "Mitochondrion" GO term in young and old, mouse CMs as observed by the GSEA pre-ranked list enrichment analysis (normalised enrichment score: -1.70; FDR q-value < 0.05). Alongside this is a column clustered heatmap displaying a list of genes from the electron transport chain (ETC) GO ontology. In both instances, genes are by column and samples by row with the colour intensity representing column Z-score, where red indicates highly and blue lowly expressed.Real-time PCR gene expression analysis of MAO-A, MnSOD and catalase in isolated mouse CMs from young (3 months) and old (20 months) mice.Mean % of 4-HNE- (top) or 8oxodG- (bottom) positive CMs from 3 month (young) or 30 month (old) aged mice. Data are mean ± SEM of n=4 per age group. 100 CMs were quantified per age group.Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in wild-type (control) compared to MAO-A transgenic mice with or without drinking water supplemented with 1.5g/kg/day NAC from the age of 4 to 24 weeks Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group.Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in MnSOD+/+ vs MnSOD-/+, Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group.Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in Catalase+/+ vs. Catalase-/-, Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group.Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in WT vs. POLG double mutant mice. Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group.Schematic depicting isolated mouse adult CMs isolated from 4 animals were treated with or without 100nM rotenone either in the presence of 5mM NAC or vehicle control (pre-treated for 30 minutes before rotenone treatment), for 24 h before fixation. Mean number of TAF (top graph) and mean % of TAF-positive nuclei (bottom graph). Data are mean ± SEM from 4 separate CM cultures isolated from 3 month-old mice. 50 CMs were quantified per condition.


Figure 7Examples of individual short axis cine - MR images of 3 or 20 month old mouse hearts . Ejection fraction and LV thickness was calculated based on manual measurements of Left ventricle epicardial and endocardial borders . % change in wall thickening calculation based on wall thickness at the 4 points indicated . Measurements were made in all cine slices at end diastole and end systole . Graphs representing data obtained from MRI analysis of > 7 animals per age group . Data are mean ± S . E . M . Asterisks denote a statistical significance at P < 0 . 05 using Mann Whitney U - test . Scale bars represent 5mm . Comparison between mean number of TAF per CM and CM area in 22 month old animals . Data are mean ± S . E . M . of n = 4 . > 100 CMs were quantified per mouse . Comparison between the % of p16 - or eGFP - positive CMs by RNA - in situ hybridization per plane in INK - ATTAC mice ( 28 - 29 month old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 per age group . 100 CMs were analysed per mouse . Mean number of TAF ( left graph ) and mean % of TAF - positive nuclei ( right graph ) in CMs . Data are mean ± S . E . M . of n = 6 per age group . 100 CMs were analysed per mouse . Mean CM area μm2 . Data are mean ± S . E . M . of n = 6 per age group , > 150 CMs analysed per mouse . % of fibrotic area evaluated by Sirius Red staining .

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Figure 7Examples of individual short axis cine-MR images of 3 or 20 month old mouse hearts. Ejection fraction and LV thickness was calculated based on manual measurements of Left ventricle epicardial and endocardial borders. % change in wall thickening calculation based on wall thickness at the 4 points indicated. Measurements were made in all cine slices at end diastole and end systole. Graphs representing data obtained from MRI analysis of >7 animals per age group. Data are mean ± S.E.M. Asterisks denote a statistical significance at P<0.05 using Mann Whitney U-test. Scale bars represent 5mm.Comparison between mean number of TAF per CM and CM area in 22 month old animals. Data are mean ± S.E.M. of n=4. >100 CMs were quantified per mouse.Comparison between the % of p16- or eGFP-positive CMs by RNA-in situ hybridization per plane in INK-ATTAC mice (28-29 month old) treated with vehicle or AP20187. Data are mean ± SEM of n=5 per age group. 100 CMs were analysed per mouse.Mean number of TAF (left graph) and mean % of TAF-positive nuclei (right graph) in CMs. Data are mean ± S.E.M. of n=6 per age group. 100 CMs were analysed per mouse.Mean CM area μm2. Data are mean ±S.E.M. of n=6 per age group, >150 CMs analysed per mouse.% of fibrotic area evaluated by Sirius Red staining.


Figure 8Quantification of mean number of TAF and % of TAF - positive CMs in 24 months old wild - type mice treated with vehicle or navitoclax ( 50mg / kg / day ) . Data are mean ± SEM of n = 5 - 8 mice per group . More than 100 CMs were quantified per animal . CM cross - sectional area in 24 months old wild - type mice treated with vehicle or navitoclax . ( Left ) Representative images of Sirius red staining and ( right ) % of fibrosis area in 24 month - old mice treated or not with navitoclax . MRI analysis of ejection fraction ( EF % ) in 24 month - old mice treated or not with navitoclax . Data are mean ± S . E . M of n = 6 mice per treatment group . left ventricle mass ( LVmass ) index in 24 month - old mice treated or not with navitoclax . Data are mean ± S . E . M of n = 6 mice per treatment group . % of ventricle wall thickness ( % WTC ) in 24 month - old mice treated or not with navitoclax . Data are mean ± S . E . M of n = 6 mice per treatment group . Examples of confocal microscopy images of CMs positive for CM marker troponin C ( TropC ) , EdU , Ki67 and Aurora B from navitoclax treated animals . ( Upper Right panel ) white arrows identify two nuclei in the same CM that have incorporated EdU . ( Lower Right panel ) white arrow identify EdU expressing Aurora B symmetrically between two nuclei . Scale bars represent 20µm . Quantification of EdU positive CMs ( mono - or multi - nucleated ) in vehicle and navitoclax treated animals . Data are mean ± S . E . M of n = 5 - 6 mice per group . % of Ki67 positive CMs in 29 month old INK - ATTAC mice treated with vehicle or AP20187 . Data are mean ± S . E . M of n = 4 - 6 mice per group .

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Figure 8Quantification of mean number of TAF and % of TAF-positive CMs in 24 months old wild-type mice treated with vehicle or navitoclax (50mg/kg/day). Data are mean± SEM of n=5-8 mice per group. More than 100 CMs were quantified per animal.CM cross-sectional area in 24 months old wild-type mice treated with vehicle or navitoclax.(Left) Representative images of Sirius red staining and (right) % of fibrosis area in 24 month-old mice treated or not with navitoclax.MRI analysis of ejection fraction (EF%) in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.left ventricle mass (LVmass) index in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.% of ventricle wall thickness (%WTC) in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.Examples of confocal microscopy images of CMs positive for CM marker troponin C (TropC), EdU, Ki67 and Aurora B from navitoclax treated animals. (Upper Right panel) white arrows identify two nuclei in the same CM that have incorporated EdU. (Lower Right panel) white arrow identify EdU expressing Aurora B symmetrically between two nuclei. Scale bars represent 20µm.Quantification of EdU positive CMs (mono- or multi-nucleated) in vehicle and navitoclax treated animals. Data are mean±S.E.M of n=5-6 mice per group.% of Ki67 positive CMs in 29 month old INK-ATTAC mice treated with vehicle or AP20187. Data are mean±S.E.M of n=4-6 mice per group.


Figure 1HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting . ( A ) Filter ‐ trap analysis of MG132 ‐ induced , SDS ‐ insoluble ubiquitinated aggregates generated in wild ‐ type ( WT ) , HDAC6 KO , and KO MEFs reconstituted with different HDAC6 constructs as indicated . The signal intensity from the ubiquitinimmunoblot ( bottom panel ) was quantified and presented as the average of the means from three independent experiments along with the standard deviation ( top ) . Note the significant accumulation of ubiquitin ‐ positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6 ‐ CD and ΔBUZ mutants . WT , HDAC6 KO , and HDAC6 KO MEFs stably expressing human HDAC6 ( hHDAC6 WT ) , HDAC6 CD ( catalytically inactive mutant ) , or HDAC6 ΔBUZ ( ubiquitin ‐ binding ‐ deficient mutant ) were analysed for the level of HDAC6 using an anti ‐ human HDAC6 antibody . Mouse endogenous HDAC6 ( mHDAC6 ) and actin levels were determined by each corresponding antibody . ( B ) WT and HDAC6 KO MEFs were treated with MG132 and subjected to Western blot analysis for LC3 , actin , and HDAC6 . ( C ) Cells were treated with MG132 as described under Materials and methods and immunostained with antibodies to ubiquitin ( green ) and LC3 ( red ) . Arrows indicate ubiquitin ‐ positive aggregates that colocalize with LC3 ‐ positive autophagosomes . Scale bar , 10 μm . ( D ) WT and ATG5 KO MEFs are treated with 2 . 5 μM MG132 for 1 day and incubated with normal growth media for 18 h . MEFs are immunostained with anti ‐ LC3 ( red ) and anti ‐ ubiquitin antibody ( green ) .

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Figure 1HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting. (A) Filter‐trap analysis of MG132‐induced, SDS‐insoluble ubiquitinated aggregates generated in wild‐type (WT), HDAC6 KO, and KO MEFs reconstituted with different HDAC6 constructs as indicated. The signal intensity from the ubiquitinimmunoblot (bottom panel) was quantified and presented as the average of the means from three independent experiments along with the standard deviation (top). Note the significant accumulation of ubiquitin‐positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6‐CD and ΔBUZ mutants. WT, HDAC6 KO, and HDAC6 KO MEFs stably expressing human HDAC6 (hHDAC6 WT), HDAC6 CD (catalytically inactive mutant), or HDAC6 ΔBUZ (ubiquitin‐binding‐deficient mutant) were analysed for the level of HDAC6 using an anti‐human HDAC6 antibody. Mouse endogenous HDAC6 (mHDAC6) and actin levels were determined by each corresponding antibody.(B) WT and HDAC6 KO MEFs were treated with MG132 and subjected to Western blot analysis for LC3, actin, and HDAC6.(C) Cells were treated with MG132 as described under Materials and methods and immunostained with antibodies to ubiquitin (green) and LC3 (red). Arrows indicate ubiquitin‐positive aggregates that colocalize with LC3‐positive autophagosomes. Scale bar, 10 μm.(D) WT and ATG5 KO MEFs are treated with 2.5 μM MG132 for 1 day and incubated with normal growth media for 18 h. MEFs are immunostained with anti‐LC3 (red) and anti‐ubiquitin antibody (green).


Figure 2 ( A ) Wild ‐ type and HDAC6 KO MEFs were transfected with pcDNA , pcDNA ‐ HDAC6WT , HDAC6CD , or HDAC6ΔBuz , along with mCherry ‐ GFP ‐ LC3 as indicated . Yellow signals indicate non ‐ acidic autophagosomes and red signals indicate acidic autophagolysosomes . Scale bar , 10 μm . ( B ) The total number of yellow vesicles was quantified from three independent experiments ( > 12 cells each ) and presented as the percentage of total mCherry ‐ GFP ‐ LC3 dots ( red plus yellow ) along with the standard deviation . ( C ) In vitro fusion assays . Autophagosomes ( APGs ) and lysosomes ( Lys ) purified from wild type and HDAC6 − / − MEFs were subjected to heterotypic and homotypic in vitro fusion assays ( representative fields are shown in Supplementary Figure S3 ) . Values are means + s . e . of the percentages of fusion from three independent experiments ( more than 10 images per each experiment ) . ( D ) EM images of wild ‐ type and HDAC6 KO MEFs in normal growth conditions . Yellow arrows , autophagosomes ; red arrows , autophagolysosomes ; green arrowheads , multilamellar bodies . ( E ) Quantification of autophagosomes and autophagolysosomes . The error bar represents the standard error .

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Figure 2(A) Wild‐type and HDAC6 KO MEFs were transfected with pcDNA, pcDNA‐HDAC6WT, HDAC6CD, or HDAC6ΔBuz, along with mCherry‐GFP‐LC3 as indicated. Yellow signals indicate non‐acidic autophagosomes and red signals indicate acidic autophagolysosomes. Scale bar, 10 μm.(B) The total number of yellow vesicles was quantified from three independent experiments (>12 cells each) and presented as the percentage of total mCherry‐GFP‐LC3 dots (red plus yellow) along with the standard deviation.(C)In vitro fusion assays. Autophagosomes (APGs) and lysosomes (Lys) purified from wild type and HDAC6−/− MEFs were subjected to heterotypic and homotypic in vitro fusion assays (representative fields are shown in Supplementary Figure S3). Values are means+s.e. of the percentages of fusion from three independent experiments (more than 10 images per each experiment).(D) EM images of wild‐type and HDAC6 KO MEFs in normal growth conditions. Yellow arrows, autophagosomes; red arrows, autophagolysosomes; green arrowheads, multilamellar bodies.(E) Quantification of autophagosomes and autophagolysosomes. The error bar represents the standard error.


Figure 3 ( A ) Autophagosome - lysosome fusion is analysed in wild ‐ type and HDAC6 KO MEFs with or without starvation ( 6 h ) using mCherry ‐ GFP ‐ LC3 as described in Figure 2A . ( B ) Wild ‐ type and HDAC6 KO MEFs were cultured in Hank ' s solution for 3 h followed by immunoblotting with an antibody for LC3 , HDAC6 , and GAPDH . ( 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 .

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Figure 3(A) Autophagosome-lysosome fusion is analysed in wild‐type and HDAC6 KO MEFs with or without starvation (6 h) using mCherry‐GFP‐LC3 as described in Figure 2A.(B) Wild‐type and HDAC6 KO MEFs were cultured in Hank's solution for 3 h followed by immunoblotting with an antibody for LC3, HDAC6, and GAPDH.(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.


Figure 4 ( A ) Wild ‐ type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to Lamp ‐ 1 ( a lysosome marker , red ) and ubiquitin ( green ) as indicated . F ‐ actin was detected by phalloidin ( blue ) . The arrows indicate ubiquitin ‐ positive aggregates that are surrounded by F ‐ actin and Lamp ‐ 1 . ( B , C ) Wild ‐ type and HDAC6 KO MEFs were transfected with mCherry ‐ GFP ‐ LC3 , followed by treatment with LatA ( 100 nM ) or nocodazole ( 250 nM ) for 6 h and analysed as described in Figure 3A . ( D ) Autophagosomes ( APGs ) and lysosomes ( Lys ) isolated from fed mouse hepatocytes were treated or not with latrunculin ( LatA ) as indicated , extensively washed to remove traces of the inhibitor , and then labelled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin . The number of total fusion events / total number of vesicles for each condition was as follows : 176 / 880 ; 233 / 1110 ; 84 / 930 ; and 180 / 950 . The differences with untreated samples were significant at * * P0 . 01 . ( E ) Autophagosomes ( APGs ) and lysosomes ( Lys ) isolated from fed or starved mouse hepatocytes were treated or not with latrunculin ( LatA ) as labelled and subjected to in vitro fusion assay . The differences with untreated samples were significant at * P0 . 05 . The number of total fusion events / total number of vesicles for each condition was as follows : 169 / 1250 ; 87 / 972 ; 190 / 1120 ; and 175 / 1165 . ( 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 .

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Figure 4(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to Lamp‐1 (a lysosome marker, red) and ubiquitin (green) as indicated. F‐actin was detected by phalloidin (blue). The arrows indicate ubiquitin‐positive aggregates that are surrounded by F‐actin and Lamp‐1.(B, C) Wild‐type and HDAC6 KO MEFs were transfected with mCherry‐GFP‐LC3, followed by treatment with LatA (100 nM) or nocodazole (250 nM) for 6 h and analysed as described in Figure 3A.(D) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed mouse hepatocytes were treated or not with latrunculin (LatA) as indicated, extensively washed to remove traces of the inhibitor, and then labelled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin. The number of total fusion events/total number of vesicles for each condition was as follows: 176/880; 233/1110; 84/930; and 180/950. The differences with untreated samples were significant at **P0.01.(E) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed or starved mouse hepatocytes were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at *P0.05. The number of total fusion events/total number of vesicles for each condition was as follows: 169/1250; 87/972; 190/1120; and 175/1165.(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.


Figure 5 ( A ) Biochemical characterization of autophagic compartments isolated from HDAC6 KO cells . Different subcellular fractions ( 75 μg protein ) isolated from the wild type ( WT ) were subjected to SDS - PAGE and immunoblotting for the indicated proteins . Hom , homogenate ; APG , autophagosomes ; APL , autophagolysosomes ; Lys , lysosomes . ( B ) Autophagosomes ( APGs ) and lysosomes ( Lys ) isolated from fed cells were treated or not with latrunculin ( LatA ) as labelled and subjected to in vitro fusion assay . The differences with untreated samples were significant at * * P0 . 01 . ( 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 .

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Figure 5(A) Biochemical characterization of autophagic compartments isolated from HDAC6 KO cells. Different subcellular fractions (75 μg protein) isolated from the wild type (WT) were subjected to SDS-PAGE and immunoblotting for the indicated proteins. Hom, homogenate; APG, autophagosomes; APL, autophagolysosomes; Lys, lysosomes.(B) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed cells were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at **P0.01.(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.


Figure 6 ( A ) Wild ‐ type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies against cortactin ( red ) , ubiquitin ( green ) , and phalloidin for F ‐ actin ( blue ) as indicated . The arrows indicated ubiquitin ‐ positive aggregates that were colocalized with F ‐ actin and cortactin . ( B ) Wild ‐ type MEFs were transfected with control or cortactin siRNA , treated with MG132 , and stained with antibodies for Lap ‐ 1 ( red , to label lysosome ) , or ubiquitin ( green ) and phalloidin for actin ( blue ) . Note that F ‐ actin staining at protein aggregates was lost , but lysosomes remained concentrated in cortactin knockdown cells ( arrow ) . ( C ) Wild ‐ type MEFs were transfected with control or cortactin siRNA , treated MG132 2 . 5 μM for 18 h , and subjected to filter ‐ trap assay using a ubiquitin antibody . The knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody against cortactin and GAPDH in the right panel . ( D ) U2OS cells were transfected with control siRNA and cortactin siRNA . Autophagosome - lysosome fusion was analysed with or without starvation ( 6 h ) using the mCherry ‐ GFP ‐ LC3 reporter as described in Figure 3A . ( 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 .

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Figure 6(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies against cortactin (red), ubiquitin (green), and phalloidin for F‐actin (blue) as indicated. The arrows indicated ubiquitin‐positive aggregates that were colocalized with F‐actin and cortactin.(B) Wild‐type MEFs were transfected with control or cortactin siRNA, treated with MG132, and stained with antibodies for Lap‐1 (red, to label lysosome), or ubiquitin (green) and phalloidin for actin (blue). Note that F‐actin staining at protein aggregates was lost, but lysosomes remained concentrated in cortactin knockdown cells (arrow).(C) Wild‐type MEFs were transfected with control or cortactin siRNA, treated MG132 2.5 μM for 18 h, and subjected to filter‐trap assay using a ubiquitin antibody. The knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody against cortactin and GAPDH in the right panel.(D) U2OS cells were transfected with control siRNA and cortactin siRNA. Autophagosome-lysosome fusion was analysed with or without starvation (6 h) using the mCherry‐GFP‐LC3 reporter as described in Figure 3A.(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.


Figure 7 ( A ) HDAC6 KO mice accumulate ubiquitin ‐ positive protein aggregates in the brain . The hippocampus and cerebral cortex regions from 6 ‐ month ‐ old wild ‐ type and ubiquitin KO littermates were subjected to immunostaining with a ubiquitin antibody and counterstained with hematoxylin . The red arrows indicate ubiquitin ‐ positive neuritic aggregates and black arrows indicate cytoplasmic aggregates . These ubiquitin ‐ positive structures were rarely observed in control littermates . Scale bar , 50 μm ( B ) Apoptotic cell death in the cortex and hippocampus region of HDAC6 KO mice as determined by TUNEL staining . Apoptotic cells were only observed in HDAC6 KO mice . Scale bar , 100 μm . ( C ) HDAC6 depletion in the Drosophila eye leads to ubiquitin ‐ positive pathology . Immunostaining for ubiquitin ( green ) in frontal eye sections of 1 ‐ day ‐ old ( d1 ) and 30 ‐ day ‐ old ( d30 ) fly eyes . The eyes of HDAC6 ‐ depleted flies ( GMR : GAL4 / UAS ‐ HDAC6RNAi ) developed ubiquitin ‐ positive cytoplasmic inclusions that become more prominent at day 30 ( d30 ) . Blue , DAPI . ( D ) Depletion of HDAC6 in the Drosophila eye leads to age ‐ dependent degeneration . Light micrographs ( left ) and corresponding Richardson ‐ stained frontal eye sections ( right ) of 1 ‐ day ‐ old and 30 ‐ day ‐ old fly eyes . The eyes of control flies ( GMR : GAL4 / + ) and HDAC6 ‐ depleted flies ( GMR : GAL4 / UAS ‐ HDAC6RNAi ) show normal highly organized ommatidial array at day 1 ; 30 ‐ day ‐ old control animals also show no defects , but 30 ‐ day ‐ old HDAC6 ‐ depleted flies show degeneration with disorganization of the ommatidial array and loss of normal eye architecture ( × 40 and × 80 ) .

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Figure 7(A) HDAC6 KO mice accumulate ubiquitin‐positive protein aggregates in the brain. The hippocampus and cerebral cortex regions from 6‐month‐old wild‐type and ubiquitin KO littermates were subjected to immunostaining with a ubiquitin antibody and counterstained with hematoxylin. The red arrows indicate ubiquitin‐positive neuritic aggregates and black arrows indicate cytoplasmic aggregates. These ubiquitin‐positive structures were rarely observed in control littermates. Scale bar, 50 μm(B) Apoptotic cell death in the cortex and hippocampus region of HDAC6 KO mice as determined by TUNEL staining. Apoptotic cells were only observed in HDAC6 KO mice. Scale bar, 100 μm.(C) HDAC6 depletion in the Drosophila eye leads to ubiquitin‐positive pathology. Immunostaining for ubiquitin (green) in frontal eye sections of 1‐day‐old (d1) and 30‐day‐old (d30) fly eyes. The eyes of HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) developed ubiquitin‐ positive cytoplasmic inclusions that become more prominent at day 30 (d30). Blue, DAPI.(D) Depletion of HDAC6 in the Drosophila eye leads to age‐dependent degeneration. Light micrographs (left) and corresponding Richardson‐stained frontal eye sections (right) of 1‐day‐old and 30‐day‐old fly eyes. The eyes of control flies (GMR:GAL4/+) and HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) show normal highly organized ommatidial array at day 1; 30‐day‐old control animals also show no defects, but 30‐day‐old HDAC6‐depleted flies show degeneration with disorganization of the ommatidial array and loss of normal eye architecture ( × 40 and × 80).


Figure 1A . Spinning disk time - lapse microscopy of WT and UBAP2L KO HeLa cells synchronized with double thymidine block and release ( DTBR ) in mitosis . The selected frames of the movies are depicted and the corresponding time is indicated in minutes . SiR - DNA was used for DNA staining . I , J . The time of mitotic entry ( I ) and from prophase to anaphase ( J ) was quantified . At least 50 cells per condition were analyzed for each experiment ( n = 3 ) . Red bars represent the mean . K - M . The percentages of cells with abnormal mitosis ( chromosome misalignments and / or DNA bridges ) ( K ) , polylobed or multinucleated daughter cells ( L ) and mitotic cell death ( M ) were quantified . At least 50 cells per condition were analyzed . N . The nuclear size of at least 1000 control and UBAP2L - downregulated interphasic cells was measured . Red bars represent the mean .

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Figure 1A. Spinning disk time-lapse microscopy of WT and UBAP2L KO HeLa cells synchronized with double thymidine block and release (DTBR) in mitosis. The selected frames of the movies are depicted and the corresponding time is indicated in minutes. SiR-DNA was used for DNA staining.I, J. The time of mitotic entry (I) and from prophase to anaphase (J) was quantified. At least 50 cells per condition were analyzed for each experiment (n=3). Red bars represent the mean. K-M. The percentages of cells with abnormal mitosis (chromosome misalignments and/or DNA bridges) (K), polylobed or multinucleated daughter cells (L) and mitotic cell death (M) were quantified. At least 50 cells per condition were analyzed. N. The nuclear size of at least 1000 control and UBAP2L-downregulated interphasic cells was measured. Red bars represent the mean.


Figure 2A - C . Immunofluorescence ( IF ) representative pictures of WT or UBAP2L KO cells synchronized in G1 / S with DTB or in early mitosis with DTBR ( A ) and quantification of the percentage of cells with normal ( 2 ) or abnormal number of centrosomes in interphase ( B ) or in early mitosis ( C ) . At least 50 cells per condition were analyzed for each experiment . Graphs represent the mean of three biological replicates ± SD ( two sample two - tailed t - test ns = non - significant ) . D - F . IF representative pictures of HeLa cells transfected with siCTL or siUBAP2L for 48h and synchronized in G1 / S with DTB or in early mitosis with DTBR ( D ) and quantification of the percentage of cells with normal ( 2 ) or abnormal number of centrosomes in interphase ( E ) or in early mitosis ( F ) . At least 50 cells per condition were analyzed for each experiment . Graphs represent the mean of three biological replicates ± SD ( two sample two - tailed t - test ns = non - significant ) . G , H . IF representative pictures of WT or UBAP2L KO cells synchronized in mitosis with DTBR ( G ) and quantification of the percentage of cells with bipolar or non - bipolar mitotic spindle ( H ) . At least 50 cells per condition were analyzed for each experiment . The graph represents the mean of three biological replicates ± SD ( two sample two - tailed t - test ns = non - significant ) .

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Figure 2A-C. Immunofluorescence (IF) representative pictures of WT or UBAP2L KO cells synchronized in G1/S with DTB or in early mitosis with DTBR (A) and quantification of the percentage of cells with normal (2) or abnormal number of centrosomes in interphase (B) or in early mitosis (C). At least 50 cells per condition were analyzed for each experiment. Graphs represent the mean of three biological replicates ± SD (two sample two-tailed t-test ns=non-significant).D-F. IF representative pictures of HeLa cells transfected with siCTL or siUBAP2L for 48h and synchronized in G1/S with DTB or in early mitosis with DTBR (D) and quantification of the percentage of cells with normal (2) or abnormal number of centrosomes in interphase (E) or in early mitosis (F). At least 50 cells per condition were analyzed for each experiment. Graphs represent the mean of three biological replicates ± SD (two sample two-tailed t-test ns=non-significant).G, H. IF representative pictures of WT or UBAP2L KO cells synchronized in mitosis with DTBR (G) and quantification of the percentage of cells with bipolar or non-bipolar mitotic spindle (H). At least 50 cells per condition were analyzed for each experiment. The graph represents the mean of three biological replicates ± SD (two sample two-tailed t-test ns=non-significant).


Figure 3D . WB analysis of WT or UBAP2L KO HeLa cell lysates from G1 / S synchronized cells using DTB . Proteins MW is indicated in kDa . WB is representative of three independent replicates . E , F . IF representative pictures of G1 / S synchronized WT or UBAP2L KO HeLa cells using DTB and quantification of PLK1 nuclear intensity ( A . U . ) ROIs are shown in the corresponding numbered panels . Scale bar , 5µm . At least 250 cells were quantified per condition for each replicate . Each dot of graphs ( F ) represents PLK1 nuclear intensity in a single nucleus where PLK1 signal was detectable . Interphasic cells which do not express PLK1 were excluded from the quantification . The measurements of four biological replicates are combined , red bars represent the mean ( Kruskal - Wallis test with Dunn ' s correction * * P < 0 , 01 , * * * * P < 0 , 0001 ) . H . WB analysis of HeLa cell lysates from unsynchronized cells treated with the indicated siRNAs . Proteins MW is indicated in kDa . WB is representative of three independent replicates . I . Quantification of the relative protein levels of BubR1 . Graphs represent the average ratio of BubR1 ( lower band ) or phospho - BubR1 ( upper band ) / GAPDH ( A . U . ) from three biological replicates ± SD ( two sample two - tailed t - test * P < 0 , 05 , ns = non - significant ) .

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Figure 3D. WB analysis of WT or UBAP2L KO HeLa cell lysates from G1/S synchronized cells using DTB. Proteins MW is indicated in kDa. WB is representative of three independent replicates.E, F. IF representative pictures of G1/S synchronized WT or UBAP2L KO HeLa cells using DTB and quantification of PLK1 nuclear intensity (A.U.) ROIs are shown in the corresponding numbered panels. Scale bar, 5µm. At least 250 cells were quantified per condition for each replicate. Each dot of graphs (F) represents PLK1 nuclear intensity in a single nucleus where PLK1 signal was detectable. Interphasic cells which do not express PLK1 were excluded from the quantification. The measurements of four biological replicates are combined, red bars represent the mean (Kruskal-Wallis test with Dunn's correction **P<0,01, ****P<0,0001).H. WB analysis of HeLa cell lysates from unsynchronized cells treated with the indicated siRNAs. Proteins MW is indicated in kDa. WB is representative of three independent replicates. I. Quantification of the relative protein levels of BubR1. Graphs represent the average ratio of BubR1 (lower band) or phospho-BubR1 (upper band)/GAPDH (A.U.) from three biological replicates ± SD (two sample two-tailed t-test *P<0,05, ns=non-significant).


Figure 4B . WB analysis of control or UBAP2L - silenced HeLa lysates from mitotic cells synchronized with 1µM paclitaxel and treated with 100µg / mL CHX for the indicated times . Proteins MW is indicated in kDa . WB is representative of three independent replicates . C . WB analysis of WT or UBAP2L KO HeLa lysates from G1 / S synchronized ( DTB ) cells treated with 25µM of the proteasomal inhibitor MG132 for the indicated times . Proteins MW is indicated in kDa . WB is representative of three independent replicates . D . Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in G1 / S using DTB , in S using 2mM hydroxyurea ( HU ) for 20h or in G2 using 10µM of CDK1 inhibitor RO 3306 for 20h .

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Figure 4B. WB analysis of control or UBAP2L-silenced HeLa lysates from mitotic cells synchronized with 1µM paclitaxel and treated with 100µg/mL CHX for the indicated times. Proteins MW is indicated in kDa. WB is representative of three independent replicates.C. WB analysis of WT or UBAP2L KO HeLa lysates from G1/S synchronized (DTB) cells treated with 25µM of the proteasomal inhibitor MG132 for the indicated times. Proteins MW is indicated in kDa. WB is representative of three independent replicates.D. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in G1/S using DTB, in S using 2mM hydroxyurea (HU) for 20h or in G2 using 10µM of CDK1 inhibitor RO 3306 for 20h.


Figure 5IF analysis of G1 / S synchronized WT or UBAP2L KO HeLa cells using DTB and transfected with the indicated flag - tagged UBAP2L protein fragments for 48h ( A ) Scale bar , 5µm . At least 100 cells per condition were quantified for each experiment . Graphs represent the mean of three replicates ± SD ( one - way ANOVA with Sidak ' s correction * P < 0 , 05 , * * P < 0 , 01 , ns = non - significant ) . IF representative pictures of WT or UBAP2L KO HeLa cells transfected with the indicated flag - tagged UBAP2L protein fragments for 48h and synchronized in mitosis using monastrol release ( MR ) ( C ) Scale bar , 5µm . At least 50 cells per condition were quantified for each experiment . Colony formation assay of WT or UBAP2L KO HeLa cells transiently transfected with the indicated flag - tagged UBAP2L protein fragments after 7 days of culture . Graphs represent the mean of three replicates ± SD ( one - way ANOVA with Sidak ' s correction * P < 0 , 05 , * * * * P < 0 , 0001 , ns = non - significant ) .

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Figure 5IF analysis of G1/S synchronized WT or UBAP2L KO HeLa cells using DTB and transfected with the indicated flag-tagged UBAP2L protein fragments for 48h (A) Scale bar, 5µm. At least 100 cells per condition were quantified for each experiment. Graphs represent the mean of three replicates ± SD (one-way ANOVA with Sidak's correction *P<0,05, **P<0,01, ns=non-significant).IF representative pictures of WT or UBAP2L KO HeLa cells transfected with the indicated flag-tagged UBAP2L protein fragments for 48h and synchronized in mitosis using monastrol release (MR) (C) Scale bar, 5µm. At least 50 cells per condition were quantified for each experiment.Colony formation assay of WT or UBAP2L KO HeLa cells transiently transfected with the indicated flag-tagged UBAP2L protein fragments after 7 days of culture. Graphs represent the mean of three replicates ± SD (one-way ANOVA with Sidak's correction *P<0,05, ****P<0,0001, ns=non-significant).


Figure 6Representative IF images of WT or UBAP2L KO HeLa cells synchronized in G1 / S using DTB and transfected with the indicated flag - tagged UBAP2L constructs and control or G3BP1 / 2 siRNAs for 48h ( A ) Representative IF images of WT or UBAP2L KO HeLa cells synchronized in G1 / S using DTB and treated with vehicle ( DMSO ) or 25µM of MG132 for 4h ( C )

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Figure 6Representative IF images of WT or UBAP2L KO HeLa cells synchronized in G1/S using DTB and transfected with the indicated flag-tagged UBAP2L constructs and control or G3BP1/2 siRNAs for 48h (A)Representative IF images of WT or UBAP2L KO HeLa cells synchronized in G1/S using DTB and treated with vehicle (DMSO) or 25µM of MG132 for 4h (C)


Figure 7Representative IF images of control or UBAP2L - downregulated cells synchronized in prometaphase using 1mM monastrol and released at the indicated time points . E . Spinning disk time - lapse microscopy of PLK1 - eGFP Knock - In ( KI ) HeLa cells synchronized with DTBR in mitosis . The selected frames of the movies are depicted and the corresponding time is indicated in minutes . SiR - DNA was used for DNA staining .

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Figure 7Representative IF images of control or UBAP2L-downregulated cells synchronized in prometaphase using 1mM monastrol and released at the indicated time points.E. Spinning disk time-lapse microscopy of PLK1-eGFP Knock-In (KI) HeLa cells synchronized with DTBR in mitosis. The selected frames of the movies are depicted and the corresponding time is indicated in minutes. SiR-DNA was used for DNA staining.


Figure 8A , B . Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR ( A ) and quantification of the relative PLK1 intensity at the inner centromere ( A . U . ) ( B ) . ROIs are shown in the corresponding numbered panels . Scale bar , 5µm . At least 50 cells were quantified per condition for each experiment . Each dot represents PLK1 / INCENP intensity ratio at a single centromere . The measurements of three biological replicates are combined , red bars represent the mean ( Mann - Whitney test * * * * P < 0 , 0001 ) . C , D . Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR ( C ) and quantification of the relative PLK1 intensity at the centrosomes ( A . U . ) ( D ) . ROIs are shown in the corresponding numbered panels . Scale bar , 5µm . At least 50 cells were quantified per condition for each experiment . Each dot represents PLK1 / Pericentrin intensity ratio at a single centrosome . The measurements of three biological replicates are combined , red bars represent the mean ( Mann - Whitney test * * * * P < 0 , 0001 ) . E , F . Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR ( E ) and quantification of the relative PLK1 intensity at the midbody ( A . U . ) ( F ) . ROIs are shown in the corresponding numbered panels . Scale bar , 4µm . At least 50 cells were quantified per condition for each experiment . Each dot represents PLK1 / PRC1 intensity ratio at the midbody . The measurements of three biological replicates are combined , red bars represent the mean ( Mann - Whitney test * * P < 0 , 01 ) . G . Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR ( n = 3 ) to qualitatively assess the localization of PLK1 at the mitotic spindle with α - tubulin staining . ROIs are shown in the corresponding numbered panels . Scale bar , 5µm .

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Figure 8A, B. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (A) and quantification of the relative PLK1 intensity at the inner centromere (A.U.) (B). ROIs are shown in the corresponding numbered panels. Scale bar, 5µm. At least 50 cells were quantified per condition for each experiment. Each dot represents PLK1/INCENP intensity ratio at a single centromere. The measurements of three biological replicates are combined, red bars represent the mean (Mann-Whitney test ****P<0,0001).C, D. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (C) and quantification of the relative PLK1 intensity at the centrosomes (A.U.) (D). ROIs are shown in the corresponding numbered panels. Scale bar, 5µm. At least 50 cells were quantified per condition for each experiment. Each dot represents PLK1/Pericentrin intensity ratio at a single centrosome. The measurements of three biological replicates are combined, red bars represent the mean (Mann-Whitney test ****P<0,0001).E, F. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (E) and quantification of the relative PLK1 intensity at the midbody (A.U.) (F). ROIs are shown in the corresponding numbered panels. Scale bar, 4µm. At least 50 cells were quantified per condition for each experiment. Each dot represents PLK1/PRC1 intensity ratio at the midbody. The measurements of three biological replicates are combined, red bars represent the mean (Mann-Whitney test **P<0,01).G. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (n=3) to qualitatively assess the localization of PLK1 at the mitotic spindle with α-tubulin staining. ROIs are shown in the corresponding numbered panels. Scale bar, 5µm.


Figure 9D . WB analysis of control ( siCTL ) or siUBAP2L 48h - treated HeLa cells were synchronized with 1mM monastrol for 16h , treated with DMSO or with 10nM of the PLK1 inhibitor BI2536 for 45min , subsequently washed out from monastrol for the indicated time and collected for protein extraction . This moderate treatment is sufficient to restore the aberrant PLK1 catalytic activity observed in UBAP2L KO cells to the levels of the WT , enabling correct mitotic progression but prevents control cells to progress through mitosis . Proteins MW is indicated in kDa . WB is representative of three independent replicates . DAPI staining of the experiment described in ( D ) showing different mitotic stages ( E ) .

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Figure 9D. WB analysis of control (siCTL) or siUBAP2L 48h-treated HeLa cells were synchronized with 1mM monastrol for 16h, treated with DMSO or with 10nM of the PLK1 inhibitor BI2536 for 45min, subsequently washed out from monastrol for the indicated time and collected for protein extraction. This moderate treatment is sufficient to restore the aberrant PLK1 catalytic activity observed in UBAP2L KO cells to the levels of the WT, enabling correct mitotic progression but prevents control cells to progress through mitosis. Proteins MW is indicated in kDa. WB is representative of three independent replicates.DAPI staining of the experiment described in (D) showing different mitotic stages (E).


Fig . 1 . ( B ) High - content screening ( HCS ) of 5 , 406 compounds in two batches in duplicate and calculation of Z ' - factors between high ( MERS - CoV infection , black ) and low ( mock , green ) values . ( C ) Correlation of duplicate screening sets . Compounds with MERS - CoV inhibition and cell viability > 70 % were regarded as primary hits . Scatter plot shows MERS - CoV inhibition ratios overlaid with cell viability ratios .

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Fig. 1.(B) High-content screening (HCS) of 5,406 compounds in two batches in duplicate and calculation of Z'-factors between high (MERS-CoV infection, black) and low (mock, green) values.(C) Correlation of duplicate screening sets. Compounds with MERS-CoV inhibition and cell viability >70% were regarded as primary hits. Scatter plot shows MERS-CoV inhibition ratios overlaid with cell viability ratios.


Fig . 3 . Time - of - addition study with selected FDA - approved drugs . Five FDA - approved drugs were analyzed by time - course experiments to determine the stage in the MERS - CoV life cycle that is inhibited by the drugs . Vero cells were infected at a multiplicity of infection ( MOI ) of 5 with MERS - CoV , and FDA - approved drugs were administered at six - time points prior to or post - infection as indicated . Drug concentrations were higher than the 90 % inhibitory concentration ( IC90 ) values of the drugs , and chloroquine was used as an early stage infection inhibitor control .

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Fig. 3.Time-of-addition study with selected FDA-approved drugs.Five FDA-approved drugs were analyzed by time-course experiments to determine the stage in the MERS-CoV life cycle that is inhibited by the drugs. Vero cells were infected at a multiplicity of infection (MOI) of 5 with MERS-CoV, and FDA-approved drugs were administered at six-time points prior to or post-infection as indicated. Drug concentrations were higher than the 90% inhibitory concentration (IC90) values of the drugs, and chloroquine was used as an early stage infection inhibitor control.


Figure 1 ( A ) Immunoblot analysis with anti - LC3 antibody shows that NO donors reduced LC3 - II levels in rat primary cortical neurons and decreased autophagosome synthesis in bafilomycin A1 - treated rat primary cortical neurons and HeLa cells . ( B - E ) Confocal microscopy images and analysis of autophagic flux by automated Cellomics microscopy show that NO donors reduced autolysosome numbers in mRFP - GFP - LC3 HeLa cells grown in full medium ( FM ) ( B and D ) or starved with HBSS ( C and E ) . Cells in HBSS had more autolysosomes compared to FM ( C ) . Autolysosome number in untreated cells in FM is set at 100 % . ( F ) NO donors increased EGFP - HDQ74 aggregates in a dose - dependent manner in EGFP - HDQ74 - transfected Atg5 + / + , but not in Atg5 − / − , MEFs . Atg5 − / − MEFs had more EGFP - HDQ74 aggregates than Atg5 + / + MEFs . ( G and H ) Confocal microscopy images ( H ) and immunofluorescence analysis with anti - Atg16 antibody ( G ) show that NO donors reduced the percentage of HeLa cells with Atg16 - positive structures ( arrows ) under starvation . Graphical data denote mean ± SEM .

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Figure 1(A) Immunoblot analysis with anti-LC3 antibody shows that NO donors reduced LC3-II levels in rat primary cortical neurons and decreased autophagosome synthesis in bafilomycin A1-treated rat primary cortical neurons and HeLa cells.(B-E) Confocal microscopy images and analysis of autophagic flux by automated Cellomics microscopy show that NO donors reduced autolysosome numbers in mRFP-GFP-LC3 HeLa cells grown in full medium (FM) (B and D) or starved with HBSS (C and E). Cells in HBSS had more autolysosomes compared to FM (C). Autolysosome number in untreated cells in FM is set at 100%.(F) NO donors increased EGFP-HDQ74 aggregates in a dose-dependent manner in EGFP-HDQ74-transfected Atg5+/+, but not in Atg5−/−, MEFs. Atg5−/− MEFs had more EGFP-HDQ74 aggregates than Atg5+/+ MEFs.(G and H) Confocal microscopy images (H) and immunofluorescence analysis with anti-Atg16 antibody (G) show that NO donors reduced the percentage of HeLa cells with Atg16-positive structures (arrows) under starvation. Graphical data denote mean ± SEM.


Figure 2 ( A ) NO donors reduced JNK1 phosphorylation in HeLa cells , as analyzed by immunoprecipitation with anti - JNK1 agarose - conjugated beads and immunoblotting with anti - phospho - JNK antibody . ( B ) NO donors reduced Bcl - 2 phosphorylation in HeLa cells , as analyzed by immunoblotting with anti - phospho - Bcl - 2 antibody . C ) Immunoblot analysis with anti - phospho - Bcl - 2 antibody shows that DETA NONOate could not reduce phospho - Bcl - 2 in Flag - CA JNK1 - transfected HeLa cells , where phospho - Bcl - 2 was higher compared to mock - transfected cells . ( D and E ) Immunoprecipitation with anti - Flag M2 affinity agarose gel and immunoblotting with anti - Myc antibody shows that NO donors increased the interaction of Flag - Beclin 1 with WT Myc - Bcl - 2 ( D ) , but not with AAA Myc - Bcl - 2 ( E ) , in HeLa cells transfected with WT Myc - Bcl - 2 ( D ) or AAA Myc - Bcl - 2 ( E ) along with either empty Flag or Flag - Beclin 1 . Asterisk denotes IgG band ( E ) . ( F ) Immunoprecipitation with anti - Flag M2 affinity agarose gel and immunoblotting with anti - Vps34 antibody shows that NO donors decreased Flag - Beclin 1 - hVps34 interaction in HeLa cells transfected with hVps34 along with either empty Flag or Flag - Beclin 1 . ( G ) Immunoprecipitation with anti - Flag M2 affinity agarose gel and immunoblotting with anti - Vps34 antibody shows that AAA Myc - Bcl - 2 decreased Flag - Beclin 1 - hVps34 interaction in the presence ( right panel ) or absence ( left panel ) of DETA NONOate in HeLa cells transfected with hVps34 along with either empty Flag or Flag - Beclin 1 , and with pcDNA3 . 1 or AAA Myc - Bcl - 2 . Graphical data denote mean ± SEM .

O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) NO donors reduced JNK1 phosphorylation in HeLa cells, as analyzed by immunoprecipitation with anti-JNK1 agarose-conjugated beads and immunoblotting with anti-phospho-JNK antibody.(B) NO donors reduced Bcl-2 phosphorylation in HeLa cells, as analyzed by immunoblotting with anti-phospho-Bcl-2 antibody.C) Immunoblot analysis with anti-phospho-Bcl-2 antibody shows that DETA NONOate could not reduce phospho-Bcl-2 in Flag-CA JNK1-transfected HeLa cells, where phospho-Bcl-2 was higher compared to mock-transfected cells.(D and E) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Myc antibody shows that NO donors increased the interaction of Flag-Beclin 1 with WT Myc-Bcl-2 (D), but not with AAA Myc-Bcl-2 (E), in HeLa cells transfected with WT Myc-Bcl-2 (D) or AAA Myc-Bcl-2 (E) along with either empty Flag or Flag-Beclin 1. Asterisk denotes IgG band (E).(F) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that NO donors decreased Flag-Beclin 1-hVps34 interaction in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1.(G) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that AAA Myc-Bcl-2 decreased Flag-Beclin 1-hVps34 interaction in the presence (right panel) or absence (left panel) of DETA NONOate in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1, and with pcDNA3.1 or AAA Myc-Bcl-2. Graphical data denote mean ± SEM.


Figure 3 ( A ) Immunoblot analysis with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated WT Bcl - 2 and AAA Bcl - 2 MEFs . ( B and C ) NO donors decreased EGFP - LC3 vesicles in EGFP - LC3 - transfected WT Bcl - 2 and AAA Bcl - 2 MEFs ( C ) . Images were acquired by a confocal microscope ( B ) . WT Bcl - 2 and AAA Bcl - 2 MEFs were analyzed separately . ( D ) NO donors increased EGFP - HDQ74 aggregates in EGFP - HDQ74 - transfected WT Bcl - 2 and AAA Bcl - 2 MEFs . WT Bcl - 2 and AAA Bcl - 2 MEFs were analyzed separately . ( E ) Immunoblot analysis with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated WT Jnk , Jnk1 − / − , and Jnk2 − / − MEFs . ( F ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in HeLa cells . ( G ) Confocal microscope images of immunofluorescence with anti - phospho - S6 antibody show that DETA NONOate increased S6 phosphorylation ; arrowhead shows a cell where this effect was not observed . ( H ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in WT Bcl - 2 and AAA Bcl - 2 MEFs . ( I ) Confocal microscope images of immunofluorescence with anti - phospho - mTOR and anti - LAMP1 antibodies in HeLa cells show that DETA NONOate increased phospho - mTOR but did not alter its distribution with lysosomes . Graphical data denote mean ± SEM .

O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Bcl-2 and AAA Bcl-2 MEFs.(B and C) NO donors decreased EGFP-LC3 vesicles in EGFP-LC3-transfected WT Bcl-2 and AAA Bcl-2 MEFs (C). Images were acquired by a confocal microscope (B). WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately.(D) NO donors increased EGFP-HDQ74 aggregates in EGFP-HDQ74-transfected WT Bcl-2 and AAA Bcl-2 MEFs. WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately.(E) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Jnk, Jnk1−/−, and Jnk2−/− MEFs.(F) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in HeLa cells.(G) Confocal microscope images of immunofluorescence with anti-phospho-S6 antibody show that DETA NONOate increased S6 phosphorylation; arrowhead shows a cell where this effect was not observed.(H) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in WT Bcl-2 and AAA Bcl-2 MEFs.(I) Confocal microscope images of immunofluorescence with anti-phospho-mTOR and anti-LAMP1 antibodies in HeLa cells show that DETA NONOate increased phospho-mTOR but did not alter its distribution with lysosomes. Graphical data denote mean ± SEM.


Figure 4 ( A ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in Tsc2 + / + MEFs , but not in Tsc2 − / − , MEFs . ( B ) NO donors reduced phosphorylation of IKKα / β and AMPKα in MEFs , as analyzed by immunoblotting with anti - phospho - IKKα / β and anti - phospho - AMPKα antibodies , respectively . ( C ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in Ikkβ + / + MEFs , but not in Ikkβ − / − MEFs . ( D ) Immunoblot analysis with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated Tsc2 + / + and Tsc2 − / − MEFs . ( E ) DETA NONOate decreased EGFP - LC3 vesicles in Tsc2 + / + and Tsc2 − / − MEFs transfected with EGFP - LC3 , along with either pcDNA3 . 1 or AAA Myc - Bcl - 2 . Although it further reduced EGFP - LC3 vesicles in Tsc2 + / + MEFs expressing AAA Myc - Bcl - 2 compared to mock - transfected Tsc2 + / + MEFs , this effect was not seen in Tsc2 − / − MEFs . Tsc2 + / + and Tsc2 − / − MEFs were analyzed separately . Graphical data denote mean ± SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Tsc2+/+ MEFs, but not in Tsc2−/−, MEFs.(B) NO donors reduced phosphorylation of IKKα/β and AMPKα in MEFs, as analyzed by immunoblotting with anti-phospho-IKKα/β and anti-phospho-AMPKα antibodies, respectively.(C) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Ikkβ+/+ MEFs, but not in Ikkβ−/− MEFs.(D) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated Tsc2+/+ and Tsc2−/− MEFs.(E) DETA NONOate decreased EGFP-LC3 vesicles in Tsc2+/+ and Tsc2−/−MEFs transfected with EGFP-LC3, along with either pcDNA3.1 or AAA Myc-Bcl-2. Although it further reduced EGFP-LC3 vesicles in Tsc2+/+MEFs expressing AAA Myc-Bcl-2 compared to mock-transfected Tsc2+/+MEFs, this effect was not seen in Tsc2−/−MEFs. Tsc2+/+ and Tsc2−/−MEFs were analyzed separately. Graphical data denote mean ± SEM.


Figure 5 ( A - D ) Immunoblot analyses with anti - LC3 , along with anti - nNOS , anti - iNOS , and anti - eNOS antibodies , show decreased LC3 - II levels in stable HEK293 cell lines overexpressing nNOS ( A and D ) , iNOS - GFP ( B and D ) , or eNOS ( C and D ) compared to HEK293 control cells in the presence or absence of bafilomycin A1 . ( E and F ) mCherry - LC3 vesicles were reduced in mCherry - LC3 - transfected stable HEK293 NOS cell lines compared to HEK293 control cells cultured in HBSS , an effect that was restored in the NOS cells by L - NAME ( F ) . Images were acquired by a confocal microscope ( E ) . ( G and H ) Confocal microscopy images ( G ) and immunofluorescence analysis with anti - Atg16 antibody show that stable HEK293 NOS cell lines had fewer Atg16 - positive structures under starvation , compared to HEK293 control cells ( H ) . Graphical data denote mean ± SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-D) Immunoblot analyses with anti-LC3, along with anti-nNOS, anti-iNOS, and anti-eNOS antibodies, show decreased LC3-II levels in stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and D), or eNOS (C and D) compared to HEK293 control cells in the presence or absence of bafilomycin A1.(E and F) mCherry-LC3 vesicles were reduced in mCherry-LC3-transfected stable HEK293 NOS cell lines compared to HEK293 control cells cultured in HBSS, an effect that was restored in the NOS cells by L-NAME (F). Images were acquired by a confocal microscope (E).(G and H) Confocal microscopy images (G) and immunofluorescence analysis with anti-Atg16 antibody show that stable HEK293 NOS cell lines had fewer Atg16-positive structures under starvation, compared to HEK293 control cells (H). Graphical data denote mean ± SEM.


Figure 6 ( A - F ) Immunoblot analyses with anti - nNOS , anti - iNOS , anti - eNOS , anti - phospho - JNK1 , anti - phospho - Bcl - 2 , anti - phospho - S6K , and anti - phospho - S6 antibodies show that stable HEK293 cell lines overexpressing nNOS ( A and D ) , iNOS - GFP ( B and E ) , or eNOS ( C and F ) inhibited both the JNK1 - Bcl - 2 and mTORC1 pathways compared to the HEK293 control cells under basal ( FM ) ( A - C ) or starvation ( HBSS ) ( D - F ) conditions . Graphical data denote mean ± SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A-F) Immunoblot analyses with anti-nNOS, anti-iNOS, anti-eNOS, anti-phospho-JNK1, anti-phospho-Bcl-2, anti-phospho-S6K, and anti-phospho-S6 antibodies show that stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and E), or eNOS (C and F) inhibited both the JNK1-Bcl-2 and mTORC1 pathways compared to the HEK293 control cells under basal (FM) (A-C) or starvation (HBSS) (D-F) conditions. Graphical data denote mean ± SEM.


Figure 7 ( A ) Immunoblot analyses with anti - LC3 antibody show that rapamycin ( 8 hr ) and L - NAME ( 24 hr ) increased autophagosome synthesis in bafilomycin A1 - treated mouse primary cortical neurons and HeLa cells . ( B and C ) Confocal microscopy images ( B ) and analysis of autophagic flux by automated Cellomics microscope ( C ) show that L - NAME increased autolysosomes in mRFP - GFP - LC3 HeLa cells . ( D ) L - NAME reduced EGFP - HDQ74 aggregates in EGFP - HDQ74 - transfected Atg5 + / + , but not in Atg5 − / − , MEFs . ( E ) Drosophila expressing mutant huntingtin exon 1 ( Q120 ) shows a significant decrease in neurodegeneration ( p < 0 . 001 , paired t test ) upon L - NAME treatment compared to DMSO . ( F ) Expression of an endogenous negative regulator of NOS activity ( NOS4 ) significantly attenuates neurodegeneration ( p < 0 . 05 , paired t test ) in Drosophila expressing mutant huntingtin exon 1 ( Q120 ) . ( G - I ) Images from the retina of transgenic HD zebrafish show mutant huntingtin aggregates ( arrows ) ( G ) . Treatment with rapamycin or L - NAME reduced the number of aggregates ( H ) . L - NAME did not reduce aggregates in the presence of NH4Cl , which increased aggregate count ( I ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Immunoblot analyses with anti-LC3 antibody show that rapamycin (8 hr) and L-NAME (24 hr) increased autophagosome synthesis in bafilomycin A1-treated mouse primary cortical neurons and HeLa cells.(B and C) Confocal microscopy images (B) and analysis of autophagic flux by automated Cellomics microscope (C) show that L-NAME increased autolysosomes in mRFP-GFP-LC3 HeLa cells.(D) L-NAME reduced EGFP-HDQ74 aggregates in EGFP-HDQ74-transfected Atg5+/+, but not in Atg5−/−, MEFs.(E) Drosophila expressing mutant huntingtin exon 1 (Q120) shows a significant decrease in neurodegeneration (p < 0.001, paired t test) upon L-NAME treatment compared to DMSO.(F) Expression of an endogenous negative regulator of NOS activity (NOS4) significantly attenuates neurodegeneration (p < 0.05, paired t test) in Drosophila expressing mutant huntingtin exon 1 (Q120).(G-I) Images from the retina of transgenic HD zebrafish show mutant huntingtin aggregates (arrows) (G). Treatment with rapamycin or L-NAME reduced the number of aggregates (H). L-NAME did not reduce aggregates in the presence of NH4Cl, which increased aggregate count (I).


Figure 1Expression profile of zRbm14a during early embryonic development . Lysates from 5 embryos were loaded in each lane and subjected to immunoblotting ( B ) . α - tubulin served as an internal control . The relative levels of zRbm14a ( B , C ) are shown as band intensities normalized to those of α - tubulin . Quantification results from three independent experiments are shown in ( C ) . Subcellular localizations of zRbm14a . The representative confocal micrographs ( D ) were acquired from the animal pole of zebrafish embryos . Exposures were kept the same during the imaging for comparison . DAPI labels nuclear DNA . Framed regions were magnified to show details . Arrows denote representative cytoplasmic puncta , whereas arrowheads point to perinuclear condensates . Subcellular localizations of zRbm14a . Subcellular localizations ( E ) were scored from the indicated number of cells from 4 to 6 embryos . F . Spindle - pole enrichment of zRbm14a condensates ( arrowheads ) in mitotic cells . Exposures were kept the same during the imaging for comparison . G . zRbm14a condensates colocalized with γ - tubulin puncta . The framed blastomeres at the indicated cell cycle stages were magnified to show details . Arrows point to spindle poles . H . Subcellular localizations of zRbm14b resembled those of zRbm14a . Zebrafish embryos were microinjected with 400 pg of GFP - Rbm14b mRNA per egg at the 1 - cell stage and fixed at 4 hpf . I . Asymmetric segregations of zRbm14a condensates in mitotic blastomeres by associating with centrosomal γ - tubulin puncta ( arrows ) . A typical 8 - cell mitotic embryo was entirely represented , with the two blastomeres in the framed region magnified to show details . The cartoon inset illustrates relative positions of the three blastomeres in the 16 - cell embryo . Numbers in yellow are relative fluorescent intensities of spindle pole - associated zRbm14a condensates , smaller ones set to 1 . Refer to Movies EV1 and EV2 for 3D - reconstructed images of the two representative cells in 2 - hpf embryos . J . Quantification results on the ratio of zRbm14a condensates associated with spindle pole pairs in mitotic blastomere .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 1Expression profile of zRbm14a during early embryonic development. Lysates from 5 embryos were loaded in each lane and subjected to immunoblotting (B). α-tubulin served as an internal control. The relative levels of zRbm14a (B, C) are shown as band intensities normalized to those of α-tubulin. Quantification results from three independent experiments are shown in (C).Subcellular localizations of zRbm14a. The representative confocal micrographs (D) were acquired from the animal pole of zebrafish embryos. Exposures were kept the same during the imaging for comparison. DAPI labels nuclear DNA. Framed regions were magnified to show details. Arrows denote representative cytoplasmic puncta, whereas arrowheads point to perinuclear condensates.Subcellular localizations of zRbm14a. Subcellular localizations (E) were scored from the indicated number of cells from 4 to 6 embryos.F. Spindle-pole enrichment of zRbm14a condensates (arrowheads) in mitotic cells. Exposures were kept the same during the imaging for comparison.G. zRbm14a condensates colocalized with γ-tubulin puncta. The framed blastomeres at the indicated cell cycle stages were magnified to show details. Arrows point to spindle poles.H. Subcellular localizations of zRbm14b resembled those of zRbm14a. Zebrafish embryos were microinjected with 400 pg of GFP-Rbm14b mRNA per egg at the 1-cell stage and fixed at 4 hpf.I. Asymmetric segregations of zRbm14a condensates in mitotic blastomeres by associating with centrosomal γ-tubulin puncta (arrows). A typical 8-cell mitotic embryo was entirely represented, with the two blastomeres in the framed region magnified to show details. The cartoon inset illustrates relative positions of the three blastomeres in the 16-cell embryo. Numbers in yellow are relative fluorescent intensities of spindle pole-associated zRbm14a condensates, smaller ones set to 1. Refer to Movies EV1 and EV2 for 3D-reconstructed images of the two representative cells in 2-hpf embryos.J. Quantification results on the ratio of zRbm14a condensates associated with spindle pole pairs in mitotic blastomere.


Figure 4Phenotypes of zRbm14 morphants . Totally 8 ng of ctrl - MO , 14 - tMOs , or 14 - MOs were co - injected with 100 pg of in - vitro transcribed GFP mRNA ( as a tracer ) into 1 - cell embryos to generate control , maternal zRbm14 , and zygotic zRbm14 morphants , respectively . Representative embryos , imaged under a dissecting microscope , are shown in ( A ) Phenotypes of zRbm14 morphants . Totally 8 ng of ctrl - MO , 14 - tMOs , or 14 - MOs were co - injected with 100 pg of in - vitro transcribed GFP mRNA ( as a tracer ) into 1 - cell embryos to generate control , maternal zRbm14 , and zygotic zRbm14 morphants , respectively . phenotyping results in ( B ) . Total numbers of embryos analyzed are listed over histograms . Sample dots are included for quantification results with no error bars . Maternal zRbm14 morphants displayed reduced cell proliferation in MZT . Embryos stained with Hoechst 33342 and Pyronin Y to mark the nucleus and the cytoplasm , respectively , were imaged from the animal pole to cover a 10 - μm z - depth with a confocal microscope ( C ) . Maximum intensity - projected images ( C ) Maternal zRbm14 morphants displayed reduced cell proliferation in MZT . 30 embryos in each condition were used to quantify cell number ( D ) , cell size ( E ) , and nuclear size ( F ) . Each sample dot represents the average value from each embryo .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Phenotypes of zRbm14 morphants. Totally 8 ng of ctrl-MO, 14-tMOs, or 14-MOs were co-injected with 100 pg of in-vitro transcribed GFP mRNA (as a tracer) into 1-cell embryos to generate control, maternal zRbm14, and zygotic zRbm14 morphants, respectively. Representative embryos, imaged under a dissecting microscope, are shown in (A)Phenotypes of zRbm14 morphants. Totally 8 ng of ctrl-MO, 14-tMOs, or 14-MOs were co-injected with 100 pg of in-vitro transcribed GFP mRNA (as a tracer) into 1-cell embryos to generate control, maternal zRbm14, and zygotic zRbm14 morphants, respectively. phenotyping results in (B). Total numbers of embryos analyzed are listed over histograms. Sample dots are included for quantification results with no error bars.Maternal zRbm14 morphants displayed reduced cell proliferation in MZT. Embryos stained with Hoechst 33342 and Pyronin Y to mark the nucleus and the cytoplasm, respectively, were imaged from the animal pole to cover a 10-μm z-depth with a confocal microscope (C). Maximum intensity-projected images (C)Maternal zRbm14 morphants displayed reduced cell proliferation in MZT. 30 embryos in each condition were used to quantify cell number (D), cell size (E), and nuclear size (F). Each sample dot represents the average value from each embryo.