EMBO/SourceData huggingface dataset:
Figure 6(B, A typical example of time developments of correlation index g(r) (B) in a mESC six hours after the removal of 2i and LIF. These were obtained from the same cell.C) visualisation of the binding frequency of Nanog-EGFP per unit area (density) (C) in a mESC six hours after the removal of 2i and LIF. Colour in C corresponds to the density of Nanog dwell events (events/μm2) within 2,000 frames. These were obtained from the same cell. Arrows indicate the emergence of hotspots of functioning Nanog, exhibiting obviously higher density than the others.
Figure 7(C, D) Plots of all data of cells categorised with expression level (red, magenta, and blue) and with the mean values (green or cyan) in each parameter in each condition of Nanog-EGFP (C) or Oct4-EGFP (D). The definition of low (blue), middle (magenta), and high (red) in +2i, +LIF, and −LIF were the same as that of Fig. 3F, and those in −LIF2d were the same as B in this figure. Error bars represent standard deviations. For Nanog-EGFP, N = 20 (high), 32 (middle) and 20 (low) for +2i, 26 (high), 18 (middle) and 21 (low) for +LIF, 20 (high), 26 (middle) and 20 cells (low) for −LIF, and 20 (high), 20 (middle) and 19 cells (low) for −LIF2d. For Oct4-EGFP, N = 22 (high), 18 (middle) and 17 (low) for +2i, 17 (high) 16 (middle) and 20 (low) for +LIF, 19 (high), 19 (middle) and 20 cells (low) for −LIF, and 16 (high), 29 (middle) and 16 cells (low) for −LIF2d. Asterisks and double asterisks indicate p-values of less than 0.05 and 0.01, respectively, in Student's t-test. Exclamation marks and double exclamation marks indicate p-values of less than 0.05 and 0.01, respectively, in Mann Whitney's U-test. Note: the data obtained in −LIF2d could not simply compare with the others because the expression levels differed between them.
Figure 8(A) Correlation plots of log10g0 and koff of Nanog-EGFP (left) and Oct4-EGFP (centre) and log10ξ and koff of Oct4-EGFP (right) in 2i (top), +LIF (middle) and −LIF (bottom) conditions. Each plot indicates the average value in a single cell. Lines are fitting results with a liner function.(D) Effect of 0.5 µM TSA addition on log10g0 of Nanog-EGFP (left) and Oct4-EGFP (right) in +LIF (filled) and -LIF (opened). Error bars represent standard deviations. For Nanog-EGFP, N = 65 for +LIF -TSA, 91 for +LIF +TSA, 66 for -LIF -TSA, and 88 cells for -LIF +TSA. For Oct4-EGFP, N = 53 for +LIF -TSA, 42 for +LIF +TSA, 58 for -LIF -TSA, and 39 cells for -LIF +TSA. Exclamation marks and double exclamation marks indicate p-values of less than 0.05 and 0.01, respectively, in Mann Whitney's U-test.
Figure 3HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.Erlotinib IC50 values were quantified in RHOB-overexpressing or RHOB-depleted (D) HCC827 cells, (E) HCC2935 cells and (F) H3255 cells, as determined by an MTS assay after 72 h treatment. RHOB overexpression or inhibition was monitored by western blotting for each condition.G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI), then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed.G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.
Figure 1A-C. PTEN mRNA levels in LCM normal ovarian surface epithelium versus high-grade serous ovarian cancer (HGSOC) epithelium or normal ovarian stroma versus ovarian cancer-associated stroma. Datasets; (A) GSE40595, (B) GSE38666, (C) Epithelium only, GSE14407. Sample size (n) and p-values, (Mann-Whitney) annotated, whiskers Min-Max, line at median.F. Overall survival (% patients, months) of OC patients. Highest quartile (Q4) versus combination of quartiles 1-3 (Q1+2+3), PTEN mRNA (TCGA, OV). Median survival (40 and 44 months), sample size (n) and p-value, Log-rank test (Mantel-Cox) annotated. G. Overall survival (% patients, months) of OC patients. Highest quartile (Q4) versus combination of quartiles 1-3 (Q1+2+3), PTEN protein. Reverse Phase Protein Array Data, TCGA OV. Median survival (46 and 57 months), sample size (n) and p-value, Log-rank test (Mantel-Cox).H. Differential abundance (x, Log Ratio between conditions; y, -Log10 q-values) of Reverse Phase Protein Array data (TCGA, OV) in patient grouped by PTEN mRNA, High (Q4) vs Low (Q1). Significant, blue (-Log10 q-values >1.3); AKT signalling pathway, labelled. I. Differential abundance (x, Log Ratio between conditions; y, -Log10 q-values) of proteins in PTEN High (Q4) vs PTEN Low (Q1) protein samples. Reverse Phase Protein Array Data, TCGA OV. Significantly altered components in AKT signalling pathway labelled (-Log10 q-value> 1.3).
Figure 2B. Time series, showing a representative spheroid for each genotype, 12 hr intervals. Arrowheads, protrusions into ECM. Scale bar, 20μm. Right, cartoon of phenotype.E. Representative phase contrast images of spheroids described in (D). Outlines pseudocoloured for classification (Spherical, green; Hyper-protrusive, blue). Scale bars, 400μm or 17μm, as indicated. Magnified individual spheroids from boxed regions at indicated timepoints. Arrowheads, protrusions into ECM.
Figure 3A. Confocal images (single slice) of Trp53-/- or Trp53-/-;Pten-/- (1.15) spheroids expressing mNeonGreen-tagged (mNG) biosensors for PI(4,5)P2 (PH-PLCδ1) or PIP3 (CYTH32G/GRP1). Magnified images from boxed regions, max projection of 8 (PH-PLCδ1) or 3 (CYTH32G/GRP1) z-slices, pseudocoloured in FIRE LUT. Arrowheads: red, cell-cell contact; yellow, nucleus; green, protrusion tip. Scale bar, 7μm. Representative of 8 (Trp53-/-) or 10 (Trp53-/-;Pten-/-) spheroids imaged across n=2 independent experiments (PH-PLCδ1) and 22 (Trp53-/-) or 23 (Trp53-/-;Pten-/-) spheroids imaged across n=4 independent experiments (CYTH32G/GRP1). B. Intensity profiles for PH-PLCδ1 and PH-CYTH3 from spheroids shown in (A). Protrusions measured are annotated on images in upper panels, yellow lines. Arrowheads: red, protrusion tips.D. Western blotting and quantitation for S6RP pS235/236, S6RP, GAPDH (sample integrity control) in Trp53-/-;Pten-/- 1.15 spheroids treated with DMSO or inhibitors annotated in (B) for 2 days. Representative of n=3 independent lysate preparations. Data, mean ± SD of pS235/236:total S6RP ratio, normalised to DMSO. P-values, unpaired, 2-tailed t tests, as annotated.Quantitation of ID8 Trp53-/-;Pten-/- spheroids treated with PI3K isoform specific inhibitors: A66 (PI3Kα), AZD8186 (PI3Kβ), AS605240 (PI3Kγ) or CAL-101 (PI3Kδ), 6hr time intervals over 72 hrs. (I) Frequency of Spherical and Hyper-protrusive phenotypes. Heatmap (grayscale) - phenotype proportion (z-score) in control. Heatmap (blue-red) - log2 fold change from control. P-values, bubble size (Cochran-Mantel-Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow-Day test, Bonferroni adjustment, non-significant). N=2 independent experiments, 3-5 technical replicates/experiment.K. Confocal image of Trp53-/-;Pten-/- (1.15) spheroids stained for PI3Kβ (green), F-actin (magenta) and Hoechst (grey). Magnified images from boxed regions, pseudocoloured in inverted grayscale (F-actin) or FIRE LUT (PI3Kβ). Yellow or white/black arrowheads, enrichment of F-actin or PI3Kβ at protrusion tips respectively. Scale bar, 5μm. Representative of n=5 spheroids.L. Intensity profiles for PI3Kβ (green) and F-Actin (magenta) from spheroid in (K). Tip measured is annotated, ECM to body, yellow arrow, tip, arrowhead.
Figure 4A-B. Quantitation of ID8 Trp53-/-;Pten-/- 1.15 spheroids expressing shScramble, shArf5 or shArf6, 6hr time intervals over 72 hrs. (A) Heatmap (viridis) - area presented as mean of Z-score values, normalised to control (shScramble). (B) Frequency of Spherical and Hyper-protrusive phenotypes. Heatmap (grayscale) - phenotype proportion (z-score) in control. Heatmap (blue-red) - log2 fold change from control. P-values, bubble size (Cochran-Mantel-Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow-Day test, Bonferroni adjustment, non-significant). N=3 independent experiments, 4-5 technical replicates/experiment. C. Representative phase contrast images of spheroids described in (A-B). Outlines pseudocoloured for classification (Spherical, green; Hyper-protrusive, blue). Magnified individual spheroids from boxed regions at indicated timepoints. Arrowheads, protrusions into ECM. Scale bars, 400μm or 17μm, as indicated.Representative confocal images and intensity profiles of ID8 Trp53-/- and Trp53-/-;Pten-/- 1.15 cells expressing mNeonGreen (mNG)-tagged ARF6 (green) and F-actin (magenta) at the invasive front of wounded monolayers Scale bar, 5μm. Tips for which ARF6-mNG and F-Actin intensity profiles were measured are annotated, ECM to body, yellow arrow.Representative confocal images and intensity profiles of ID8 Trp53-/- and Trp53-/-;Pten-/- 1.15 cells expressing mNeonGreen (mNG)-tagged ARF6 (green) and F-actin (magenta) in spheroids (max. projection ~ 10 Z-slices) Scale bar, 5μm. Tips for which ARF6-mNG and F-Actin intensity profiles were measured are annotated, ECM to body, yellow arrow.Heatmap, (J) ARF6 interactors across genotypes. White to blue colour or blue to red, ARF6 interaction score, Log2Fold Student's t-test Difference in LFQ intensity compared to control ID8 Trp53-/-;Pten-/- 1.15 TurboID alone. Interactors, sorted, descending order of mean interaction. N=4 independent lysate preparations from each sub-line.
Figure 5C. Western blot, β1-integrin (ITGB1), pS473-AKT, AKT, ARF6 from deconvolved ITGB1 sgRNA-expressing cells. VCL, loading control for ITGB1, sample integrity control for other blots. Representative blots of n=3 independent lysate preparations. D. Quantitation of (C). Data, mean ± SD for pS473-AKT:total AKT band intensity ratio, total AKT or ARF6 intensity, normalised to control (sgNT ID8 Trp53-/-;Pten-/- clone 1.15) cells. P-values, unpaired, two-tailed t-test.E-F. Frequency of Spherical and Hyper-protrusive phenotypes in ID8 Trp53-/-;Pten-/- 1.15spheroids upon CRISPR-mediated KO of (E) Itgβ1 or (F) Agap1, 6hr time intervals over 72 hrs. Heatmap (grayscale) - phenotype proportion (z-score) in control (sgNT). Heatmap (blue-red) - log2 fold change from control. P-values, bubble size (Cochran-Mantel-Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow-Day test, Bonferroni adjustment, non-significant). N=3 independent experiments, 1-5 technical replicates/experiment.H. Representative confocal images of Trp53-/- and Trp53-/-;Pten-/- clone 1.15 spheroids expressing sgNT, sgAgap1 (sg3) or sgItgb1 (sg4), stained for collagen IV (grayscale) and F-Actin (magenta). Boxed areas, basement membrane region in higher magnification. Arrowheads, Collagen IV labelling that is: well-defined, green; fragmented, yellow; absent, navy. Scale bar, 53μm. I. Quantitation of (H). Collagen IV basement membrane staining as Defined, Fragmented, or Absent in spheroids set up across n=3 independent experiments, 1 technical replicate/experiment, 5-9 fields imaged per technical replicate, 365 spheroids scored in total. Data, mean ± SD of % of spheroids in each phenotype for independent experiments, with circles representing technical replicates. Unpaired t-test, p values annotated.
Figure 6C. Western blots of ID8 Trp53-/-;Pten-/- 1.15 cells expressing either sgNT or sgAgap1 (sg3) and either mNeonGreen (mNG) or CRISPR-resistant mNG-Agap1_S or -L isoforms. Blotted for ARF6, pS473-AKT, AKT, mNG, and VCL. VCL is loading control for AKT, pS473-AKT and ARF6 and sample integrity control for others. n=3 independent lysate preparations. D. Quantitation of (C). Data, mean ± SD for ARF6 and pS473/AKT band intensity ratio, normalised to sgNT. P-values, unpaired two-tailed t-test, annotated when significant.E-F. Quantitation of ID8 Trp53-/-;Pten-/- 1.15 spheroids treated with sgNT or AGAP1-targeting sg3 and expressing either mNG or mNG-fusion with either isoform of AGAP1, 6 hr time intervals over 72 hrs. (E) Heatmap (viridis) - area presented as mean of Z-score values, normalised to control (sgNT). (F) Frequency of Spherical and Hyper-protrusive phenotypes. Heatmap (grayscale) - phenotype proportion (z-score) in control. Heatmap (blue-red) - log2 fold change from control. P-values, bubble size (Cochran-Mantel-Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow-Day test, Bonferroni adjustment, non-significant). N=3 independent experiments, 5-6 technical replicates/experiment.G. Representative phase contrast images of spheroids described in (E-F). Outlines pseudocoloured for classification (Spherical, green; Hyper-protrusive, blue). Magnified individual spheroids from boxed regions at indicated timepoints. Arrowheads, protrusions into ECM. Scale bars, 400μm or 17μm, as indicated.ARF6 and AGAP1 mRNA levels in LCM normal ovarian surface epithelium versus HGSOC epithelium or normal ovarian stroma versus OC-associated stroma. Specific datasets, sample size (n) and p-values (Mann-Whitney) annotated, whiskers Min-Max, line at median.K-O. Overall survival (% patients, months; TCGA OV dataset), of patients grouped by low (M1) versus high (M2) levels, based on a median split, of (K) ARF6 mRNA, (L) AGAP1 mRNA, (M) AGAP1 Exon 14 percentage spliced in ratio (PSI), (N) combination of ARF6 and AGAP1 mRNA, or (O) combination of ARF6 mRNA and AGAP1 Ex14 PSI. Median survival, sample size (n) and p-value, Log-rank test (Mantel-Cox) annotated.
Figure 7A-B. Immunofluorescence and confocal imaging of Trp53-/-;Pten-/- 1.15 spheroids stained for α5-integrin or β1-integrin (grey or FIRE LUT), Hoechst (blue) and F-actin (magenta). Magnified images from boxed regions shown. Arrowheads, labelling at protrusion tips. Scale bars, 5 μm. Representative of n=3 spheroids imaged. (B) Intensity profiles for integrins (grey) and F-actin (magenta) from spheroids in (A). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead.C-D. Immunofluorescence and confocal imaging of Trp53-/-;Pten-/- 1.15 spheroids stained for pFAK (Y379) or pSRC Family Kinases (SFK pY416) (grey or FIRE LUT), Hoechst (blue) and F-actin (magenta). Magnified images from boxed regions shown. Arrowheads, positive staining. Scale bars, 5 μm. Representative of n=5 spheroids imaged. (D) Intensity profiles for active FAK and Src (grey) and F-actin (magenta) from spheroids in (C). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead.E-H. Representative capture ELISA graphs (E,G) and associated quantitation (F,H) for recycling of internalized cargoes between Trp53-/- versus Trp53-/-;Pten-/- cells or Trp53-/-;Pten-/- cells expressing shScramble versus shArf6 for active β1-integrin. Graphs shown are representative of n=2 (E) or n=3 (G) independent replicates. Data, mean (black square) ± SD for repeated experiments (large circles), 1-3 technical replicates/experiment/timepoint (small circles), two-tailed t-test, p-values are annotated.I-K. Overall survival (% patients, months; TCGA OV dataset) of patients grouped into combined expression based on median mRNA split. (I) Low (red line, M1) or high (blue line, M2) expression for all mRNA, control, remaining patients (green line), (J), same as (I), but CYTH2 Ex9 PSI, rather than total CYTH2. (K), as for (I), but PTEN protein levels split by quantiles (red and blue, Q1+Q2,Q3, low PTEN, green Q4, high PTEN). Median survival, sample size (n) and p-value, Log-rank test (Mantel-Cox) annotated.
Figure 1(A) Differential intron retention (IR) analysis of RNA-seq data from samples under PABPN1 knockdown (siPABPN1) and control (NC) conditions. Significantly repressed and induced intron retention events (retained introns, RIs) are indicated by blue and red dots, respectively. The threshold of IR difference (ΔIR, siPABPN1-siNC) is 0.15, and the FDR cutoff is 0.01.(C) Cumulative frequency distributions of retained introns (RIs), up-regulated (red), and down-regulated (blue) upon PABPN1 knockdown, and of all retained introns in control (NC, green) samples by their relative intronic ranks in genes.Validation of last intron retention of six representative genes in response to PABPN1 knockdown by semi-quantitative PCR in HeLa cells treated with three different NC and PABPN1 siRNAs (D).(G) Western blot validating the degradation effect of PABPN1 under the treatment of dTAG13 for 0, 3, and 6 hours.
Figure 2(B) Western blots of the C1-C4 reporters for two endogenous regulated genes (LARP7 and COPS5) and three endogenous non-regulated control genes (TDRD9, CENPV, and SMTN).(C) RT-PCR analysis of the spliced and unspliced isoforms from the C3 and C4 reporters for two endogenous regulated genes LARP7 and COPS5.(E) RT-PCR quantification of MS2 reporters of histone 3´end (left) and Malat1 3´end (right) for two PABPN1-regulated genes LARP7 and COPS5.
Figure 3(D) RNA quantification by RT-PCR for the spliced and unspliced isoforms (top) and calculated IR scores (bottom) of the reporters Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).(E) Quantification of the spliced (EEJ) over unspliced isoforms (EIJ) of the reporters in Figure 3F. Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).(G) Splicing analysis of the C3, C4, and polyA reporters in Figure 3F for endogenous genes LARP7, COPS5, and NSUN5 by RT-PCR upon PABPN1 knockdown (top) and the quantified intron retention levels (IR score) of these reporters (bottom). Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).(J) Relative ratios of the unspliced isoforms (EIJ) to the spliced isoforms (EEJ) of the last introns for LARP7, DUSP12, and BRCA1 genes in HeLa cells treated with si-PABPN1 and then rescuing PABPN1 mutants constructed in G. Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).
Figure 4Splicing analysis of NSUN5 and LARP7 reporter mutants by RT-PCR (E)Splicing analysis of NSUN5 and LARP7 reporter mutants by Western blot (F).
Figure 5(B) Volcano plots of the enriched proteins identified by PABPN1 TurboID-MS. Significantly enriched proteins are colored in orange, and a few representative proteins are highlighted with labels.(C) RT-PCR analysis of the last intron splicing of five representative PABPN1-dependent endogenous genes upon RBM26/27 knockdown with quantified IR values. Data information Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).(G) Cumulative frequency distributions of up-regulated RIs upon PABPN1 (red), RBM26&27 (blue), ZC3H3 (green), and ZFC3H1 (yellow) knockdown by their relative intronic rank in genes.(H) Western blot validation of endogenous PABPN1 interacting with the myc-tagged RBM26 and RBM27.(I) Representative immunofluorescence images showing the localization of endogenous PABPN1 relative to SC35, and the mCherry-tagged RBM26&27 relative to SRRM2 in vivo.(K-L) Western blot of endogenous PABPN1 immunoprecipitated myc-tagged RBM27 mutated proteins.
Figure 1(A) Western Blot for pUb on HeLa control and FBXO7-/- cells after treatment with AO for indicated times.(B) Quantification of pUb signal relative to loading. Western Blots run in triplicates from three independent clones. Error bars depict S.D.. Data from three biological replicates. One-way ANOVA with multiple comparisons. p(**) = 0.0011; p(****)<0.0001.(D) Western Blot for pUb on extracts from day 12 iNeuron control and FBXO7-/- cells after treatment with BafA or AO for indicated times.
Figure 2(A) 3D-SIM images of HeLa control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (DAPI), mitochondria (HSP60) and pUb. Zoom-ins of regions of interested are enlarged in the middle panel. 3D-surface renderings of insets are shown on the right. Scale bar = 5 µm or 1 µm.(B,C) Evaluation of 3D-SIM images from HeLa datasets. The changes in pUb volume and minimal distances between mitochondria and pUb after mitophagy-induction are plotted. Error bars depict S.D. from 8-14 measured cells per condition. Two-way ANOVA with multiple comparisons; p(****)<0.0001.(D) 3D-SIM images of iN day 12 control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (DAPI), mitochondria (HSP60) and pUb. Zoom-ins of regions of interest are enlarged in the middle panel. 3D-surface renderings of insets are shown on the right. Scale bar = 5 µm or 1 µm.(E,F) Evaluation of 3D-SIM images from iNeuron datasets. The changes in pUb volume and minimal distances between mitochondria and pUb after mitophagy-induction are plotted. Error bars depict S.D. from 7-14 measured cells per condition. One-way ANOVA with multiple comparisons, p(****)<0.0001.(G) Confocal images of iNeuron d12 Control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (Hoechst33342), mitochondria (HSP60) and pUb. Scale bar = 10 µm and 5 µm. (H) Evaluation of pUb volume after mitophagy induction. Error bars depict S.D. from three biological replicates. One-way ANOVA with multiple comparisons; p(****)<0.0001.