Targets from each mode of regulation ( cf . Fig 1E ) were selected for validation by Nanostring . Shown is a scatterplot of Nanostring quantification of polysome - associated mRNA vs . cytosolic mRNA log2 fold - change ( shERα vs . shCtrl ; n = 3 ) . Genes are colored according to their identified mode of regulation ( i . e . from Fig 1E ) . Confidence ellipses ( based on Nanostring data , level 0 . 7 ) are overlaid for each mode of regulation . Selected genes from each mode of regulation are indicated .

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

Targets from each mode of regulation (cf. Fig 1E) were selected for validation by Nanostring. Shown is a scatterplot of Nanostring quantification of polysome-associated mRNA vs. cytosolic mRNA log2 fold-change (shERα vs. shCtrl; n = 3). Genes are colored according to their identified mode of regulation (i.e. from Fig 1E). Confidence ellipses (based on Nanostring data, level 0.7) are overlaid for each mode of regulation. Selected genes from each mode of regulation are indicated.


Levels of indicated proteins from shERα and control BM67 cells were determined by western blotting with their quantifications . β - actin served as loading control . Graphs represent mean ± standard deviation ( n = 3 - 5 as indicated ) . Protein expression between groups were compared using two - sided paired Student ' s t tests .

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

Levels of indicated proteins from shERα and control BM67 cells were determined by western blotting with their quantifications. β-actin served as loading control. Graphs represent mean ± standard deviation (n = 3-5 as indicated). Protein expression between groups were compared using two-sided paired Student's t tests.


Immunoblotting of BM67 cell lines extract and its quantitation following incubation with 100 µg / mL cycloheximide as indicated . Graphs represent mean ± standard deviation ( n = 5 ) .

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

Immunoblotting of BM67 cell lines extract and its quantitation following incubation with 100 µg/mL cycloheximide as indicated. Graphs represent mean ± standard deviation (n = 5).


nanoCAGE sequencing was applied to determine transcription start sites in shERα BM67 cells . The number of detected transcription start sites ( peaks ) and RefSeq transcripts when sampling increasing number of RNAseq reads are indicated to evaluate the complexity of nanoCAGE RNAseq libraries .

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

nanoCAGE sequencing was applied to determine transcription start sites in shERα BM67 cells. The number of detected transcription start sites (peaks) and RefSeq transcripts when sampling increasing number of RNAseq reads are indicated to evaluate the complexity of nanoCAGE RNAseq libraries.


Boxplots for offset and non - offset mRNAs comparing 5 ' UTR weighted lengths ( log2 scale ) , GC content ( % ) , free energy ( log10 scale , kcal / mole ) . In boxplots , the solid horizontal middle line is the median ; the lower and upper box limits correspond to the first and third quartiles ; the whiskers extend from the box limits to the most extreme value but not further than + / - 1 . 5 x the inter - quartile range ( IQR ) from the box limits . The notches extend to 1 . 58 x the IQR / √ n where n is the number of data points . Data beyond the end of the whiskers are plotted as points . Wilcoxon - Mann - Whitney tests ( two - sided ) were used to assess differences between translationally offset and non - offset mRNAs . Number of transcripts harboring at least one ( dark gray ) or no ( light gray ) uORF in a strong Kozak context were visualized as a bar chart and differences were tested using one - sided Fisher ' s exact tests . A global Bonferroni adjustment was applied on p - values . Analysis was performed on nanoCAGE data from 3 replicates of shERα BM67 cells ; the numbers of transcripts in each group are indicated in the figure .

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

Boxplots for offset and non-offset mRNAs comparing 5' UTR weighted lengths (log2 scale), GC content (%), free energy (log10 scale, kcal/mole). In boxplots, the solid horizontal middle line is the median; the lower and upper box limits correspond to the first and third quartiles; the whiskers extend from the box limits to the most extreme value but not further than +/- 1.5 x the inter-quartile range (IQR) from the box limits. The notches extend to 1.58 x the IQR / √n where n is the number of data points. Data beyond the end of the whiskers are plotted as points. Wilcoxon-Mann-Whitney tests (two-sided) were used to assess differences between translationally offset and non-offset mRNAs. Number of transcripts harboring at least one (dark gray) or no (light gray) uORF in a strong Kozak context were visualized as a bar chart and differences were tested using one-sided Fisher's exact tests. A global Bonferroni adjustment was applied on p-values. Analysis was performed on nanoCAGE data from 3 replicates of shERα BM67 cells; the numbers of transcripts in each group are indicated in the figure.


Volcano plot of ERα dependent miRNA expression in BM67 cells quantified by small RNAseq . Down - and up - regulated miRNAs ( FDR < 0 . 2 and fold - change > 1 . 25 ) are colored in blue and red , respectively .

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

Volcano plot of ERα dependent miRNA expression in BM67 cells quantified by small RNAseq. Down- and up-regulated miRNAs (FDR<0.2 and fold-change > 1.25) are colored in blue and red, respectively.


Validation of miRNA expression using qPCR . Normalized expression ( mean ± standard deviation ; n = 3 ) ( B ) and expression fold change ( shERα vs . shCtrl ) obtained from small RNAseq vs . qPCR ( C ) .

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

Validation of miRNA expression using qPCR. Normalized expression (mean ± standard deviation; n = 3) (B) and expression fold change (shERα vs. shCtrl) obtained from small RNAseq vs. qPCR (C).


Percentage of miRNA among small RNAs in shERα and shCtrl BM67 cells ( mean ± standard deviation ; n = 3 ) .

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

Percentage of miRNA among small RNAs in shERα and shCtrl BM67 cells (mean ± standard deviation; n = 3).


Assessment of the relationship between presence of miRNA target sites and translational offsetting . ( E ) For visualization , mRNAs were assigned angles describing their mode of regulation such that mRNAs with congruently modulated total and polysome - associated mRNA obtain an angle of ~ 45 degrees , whereas offset mRNAs are assigned an angle of ~ 0 degrees . ( F ) The distributions ( quantified by kernel density estimation ) of such angles were then compared between mRNAs whose levels were induced upon ERα depletion , which were also targets of each suppressed miRNAs separately ( blue lines ) ; and the set of background transcripts ( mRNAs induced upon ERα depletion but not targets of suppressed miRNAs ; black ) . ( G ) For such subsets of miRNA targets ( i . e . from F ) , the relative number of offset and non - offset mRNAs ( as determined by anota2seq analysis ) was also compared to the background ( same as in F ) using Fisher ' s exact test ; and the resulting adjusted p - values ( FDRs ; across all assessed miRNAs ) were visualized as a histogram . ( H - I ) A similar assessment as in ( F - G ) but for suppressed mRNAs and induced miRNAs . ( J ) A similar assessment as in ( H ) but for randomly selected groups of 10 miRNAs ( gray lines ) compared to background ( black ) and targets of all induced miRNA ( red ; i . e . from H ) .

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

Assessment of the relationship between presence of miRNA target sites and translational offsetting. (E) For visualization, mRNAs were assigned angles describing their mode of regulation such that mRNAs with congruently modulated total and polysome-associated mRNA obtain an angle of ~45 degrees, whereas offset mRNAs are assigned an angle of ~0 degrees. (F) The distributions (quantified by kernel density estimation) of such angles were then compared between mRNAs whose levels were induced upon ERα depletion, which were also targets of each suppressed miRNAs separately (blue lines); and the set of background transcripts (mRNAs induced upon ERα depletion but not targets of suppressed miRNAs; black). (G) For such subsets of miRNA targets (i.e. from F), the relative number of offset and non-offset mRNAs (as determined by anota2seq analysis) was also compared to the background (same as in F) using Fisher's exact test; and the resulting adjusted p-values (FDRs; across all assessed miRNAs) were visualized as a histogram. (H-I) A similar assessment as in (F-G) but for suppressed mRNAs and induced miRNAs. (J) A similar assessment as in (H) but for randomly selected groups of 10 miRNAs (gray lines) compared to background (black) and targets of all induced miRNA (red; i.e. from H).


For each codon , the average frequency ( per thousand ) is compared between transcripts induced but offset vs non - offset ( i . e . abundance mode of regulation ) upon ERα depletion in BM67 cells . Codons for the same amino acid are connected by a gray line . Heatmap of standardized residuals from a chi - squared contingency table test . Shown in red and blue are cells with counts significantly higher and lower , respectively , than expected counts under the null hypothesis ( i . e . independence between mode for regulation of gene expression and codon composition ) . A correspondence analysis for average codon frequency among transcripts annotated to each GO term . Each gray dot corresponds to one GO term . The gene expression modes observed upon ERα depletion ( Fig 1E ) were then projected into the same dimensions based on the average codon frequencies of included transcripts . A correspondence analysis for codon frequency in each regulated mRNA ( from Fig 1E ) . Each dot represents one mRNA and is colored according to its mode of regulation . Codons identified as over - represented among translationally offset mRNAs are also indicated . Unsupervised clustering of gene level codon usage normalized by amino acid counts . All regulated mRNAs ( from Fig 1E ) are shown in rows and all codons in columns . Codons identified as over - represented among mRNAs whose levels were induced but offset or suppressed but offset are indicated in light and dark blue , respectively .

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

For each codon, the average frequency (per thousand) is compared between transcripts induced but offset vs non-offset (i.e. abundance mode of regulation) upon ERα depletion in BM67 cells. Codons for the same amino acid are connected by a gray line. Heatmap of standardized residuals from a chi-squared contingency table test. Shown in red and blue are cells with counts significantly higher and lower, respectively, than expected counts under the null hypothesis (i.e. independence between mode for regulation of gene expression and codon composition). A correspondence analysis for average codon frequency among transcripts annotated to each GO term. Each gray dot corresponds to one GO term. The gene expression modes observed upon ERα depletion (Fig 1E) were then projected into the same dimensions based on the average codon frequencies of included transcripts. A correspondence analysis for codon frequency in each regulated mRNA (from Fig 1E). Each dot represents one mRNA and is colored according to its mode of regulation. Codons identified as over-represented among translationally offset mRNAs are also indicated. Unsupervised clustering of gene level codon usage normalized by amino acid counts. All regulated mRNAs (from Fig 1E) are shown in rows and all codons in columns. Codons identified as over-represented among mRNAs whose levels were induced but offset or suppressed but offset are indicated in light and dark blue, respectively.


Immunoblotting of BM67 cell extract for ELP3 after stable shRNA - mediated ERα knockdown in BM67 cells . Graphs represent mean ± standard deviation of n = 3 .

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

Immunoblotting of BM67 cell extract for ELP3 after stable shRNA-mediated ERα knockdown in BM67 cells. Graphs represent mean ± standard deviation of n=3.


qPCR analysis and immunoblotting of BM67 cell extract for indicated proteins with its quantitation following deletion of Esr1 gene by CRISPR - Cas9 technology . Graphs represent mean ± standard deviation of n = 5 .

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

qPCR analysis and immunoblotting of BM67 cell extract for indicated proteins with its quantitation following deletion of Esr1 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=5.


qPCR analysis and immunoblotting of BM67 cell extract for ELP3 and DEK with its quantitation following deletion of Elp3 gene by CRISPR - Cas9 technology . Graphs represent mean ± standard deviation of n = 4 .

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

qPCR analysis and immunoblotting of BM67 cell extract for ELP3 and DEK with its quantitation following deletion of Elp3 gene by CRISPR-Cas9 technology. Graphs represent mean ± standard deviation of n=4.


Immunoblotting and qPCR analysis comparing cell extracts obtained from BM67 cells transduced with pLentiCRISPRV2 empty vector , Elp3 - null BM67 cells and Elp3 - null BM67 cells overexpressing ELP3 protein containing silent mutations in the sgRNA binding site . Graphs represent mean ± standard deviation of n = 3 - 4 .

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

Immunoblotting and qPCR analysis comparing cell extracts obtained from BM67 cells transduced with pLentiCRISPRV2 empty vector, Elp3-null BM67 cells and Elp3-null BM67 cells overexpressing ELP3 protein containing silent mutations in the sgRNA binding site. Graphs represent mean ± standard deviation of n=3-4.


Boxplots ( plotted as in Fig 3B ) of gene expression of ELP3 , ALKBH8 and CTU2 upon treatment with 17β - E2 and / or ICI - 182780 in MCF7 cells ( extracted from ( Wardell et al , 2012 ; Data Ref : McDonnell et al , 2012 ) ) . Wilcoxon - Mann - Whitney tests ( two - sided ) were used to assess differences between conditions ( n = 10 ) and a global Bonferroni adjustment was applied on p - values .

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

Boxplots (plotted as in Fig 3B) of gene expression of ELP3, ALKBH8 and CTU2 upon treatment with 17β-E2 and/or ICI-182780 in MCF7 cells (extracted from (Wardell et al, 2012; Data Ref: McDonnell et al, 2012)). Wilcoxon-Mann-Whitney tests (two-sided) were used to assess differences between conditions (n = 10) and a global Bonferroni adjustment was applied on p-values.


Immunoblotting of MCF7 cell extracts . MCF - 7 cells were grown in phenol red - free RPMI media supplemented with 5 % charcoal - stripped serum for 24 hours and then treated with 1 nM E2 and / or 100 nM ICI - 182780 for 24 hours . Graphs represent mean ± SD of n = 5 experiments . Data were analyzed using two - sided paired Student ' s t tests .

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

Immunoblotting of MCF7 cell extracts. MCF-7 cells were grown in phenol red-free RPMI media supplemented with 5% charcoal-stripped serum for 24 hours and then treated with 1 nM E2 and/or 100 nM ICI-182780 for 24 hours. Graphs represent mean ± SD of n=5 experiments. Data were analyzed using two-sided paired Student's t tests.


ChIP - seq reveals ER binding sites proximal to the ELP3 gene . Each track represents a distinct ChIP - seq dataset .

ChIP-seq reveals ER binding sites proximal to the ELP3 gene. Each track represents a distinct ChIP-seq dataset.


Immunoblotting of MCF - 7 cell extract for indicated proteins following deletion of ELP3 gene by CRISPR - Cas9 technology .

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

Immunoblotting of MCF-7 cell extract for indicated proteins following deletion of ELP3 gene by CRISPR-Cas9 technology.


Quantification of mcm5S2 - U , mcm5U and cm5U levels in ELP3 - null MCF - 7 cells and in MCF - 7 cells treated with 100 nM ICI - 182780 for 72 hours versus their respective controls by liquid chromatography - mass spectrometry ( LC - MS ) analysis . The MCF - 7 cells were grown in phenol red - free RPMI media supplemented with 5 % charcoal - stripped serum for 24 hours prior to ICI - 182780 treatment . Graphs represent mean ± SD of n = 4 . Data were analyzed using two - sided paired Student ' s t tests . Vh , Vehicle ; ICI , ICI - 182780 .

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

Quantification of mcm5S2-U, mcm5U and cm5U levels in ELP3-null MCF-7 cells and in MCF-7 cells treated with 100 nM ICI-182780 for 72 hours versus their respective controls by liquid chromatography-mass spectrometry (LC-MS) analysis. The MCF-7 cells were grown in phenol red-free RPMI media supplemented with 5% charcoal-stripped serum for 24 hours prior to ICI-182780 treatment. Graphs represent mean ± SD of n=4. Data were analyzed using two-sided paired Student's t tests. Vh, Vehicle; ICI, ICI-182780.


G . Immunostain for Ki67 in wildtype and lymphoma - bearing spleen . Representative images are shown at left . The proportion of nuclei positive for Ki67 ( proliferation index ) is quantified at right for n = 3 spleens ( T - test p = 0 . 00002331 ) .

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

G. Immunostain for Ki67 in wildtype and lymphoma-bearing spleen. Representative images are shown at left. The proportion of nuclei positive for Ki67 (proliferation index) is quantified at right for n=3 spleens(T-test p=0.00002331).


F , G ( F ) PCR for Cγ1 - Cre and wildtype Cγ1 alleles and ( G ) , wildtype , floxed or excised Blimp1 alleles in DNA from wildtype ( wt ) control C57bl / 6J cells and purified T - cells or CD45 . 2 + cells from mouse with lymphoma ( T1 , T2 ) . PCR data were confirmed in at least 3 independent tests . Lad . = ladder .

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

F, G (F) PCR for Cγ1-Cre and wildtype Cγ1 alleles and (G), wildtype, floxed or excised Blimp1 alleles in DNA from wildtype (wt) control C57bl/6J cells and purified T-cells or CD45.2+ cells from mouse with lymphoma (T1, T2). PCR data were confirmed in at least 3 independent tests. Lad. = ladder.


F . Western blot for c - MYC in whole spleen lysates from control C57bl / 6J and mPTCL mice . Quantification of c - MYC expression normalized to vinculin housekeeper gene levels ( n = 6 ) . T - test p = 0 . 0007 . Bars represent SEM .

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

F. Western blot for c-MYC in whole spleen lysates from control C57bl/6J and mPTCL mice. Quantification of c-MYC expression normalized to vinculin housekeeper gene levels (n=6). T-test p=0.0007. Bars represent SEM.


B . Western blot for phosphorylated and total ZAP70 , AKT , S6 and GSK3β in lysates from three control ( C ) C57bl / 6J and three mPTCL ( T ) spleens . Murine DLBCL cell line A20 is a positive control for constitutively active AKT signaling .

O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot for phosphorylated and total ZAP70, AKT, S6 and GSK3β in lysates from three control (C) C57bl/6J and three mPTCL (T) spleens. Murine DLBCL cell line A20 is a positive control for constitutively active AKT signaling.


H . Western blot for γH2AX , total H2AX , CHK1 and RPA32 in control C57bl / 6J and mPTCL spleens . GAPDH is a loading control . Three representative samples per group are shown in the blot ( left ) and quantification was performed on n = 6 at right . T - test p = 0 . 0798 ( CHK1 ; not statistically significant , ns ) , p = 8 . 69E - 05 ( RPA32 ) , p = 2 . 11E - 03 ( γH2AX ) . * * * p < 0 . 001 .

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

H. Western blot for γH2AX, total H2AX, CHK1 and RPA32 in control C57bl/6J and mPTCL spleens. GAPDH is a loading control. Three representative samples per group are shown in the blot (left) and quantification was performed on n=6 at right. T-test p=0.0798 (CHK1; not statistically significant, ns), p=8.69E-05 (RPA32), p=2.11E-03 (γH2AX). ***p<0.001.


A . Sanger screen IC50 drug sensitivity data ( IC50 values ) for DDR inhibitors targeting ATR , CHK1 / 2 and Wee1 in T - ALL , PTCL ( DEL , KARPAS - 299 , SUP - M2 ) and pan - cancer cell lines . ANOVA p = 0 . 413 ( AZD6738 , ATR inhibitor ) , p = 0 . 750 ( AZD772 , CHK1 / 2 inhibitor ) , p = 0 . 119 ( MK - 8776 , ATR inhibitor ) and 0 . 282 ( AZD1775 , Wee1 inhibitor ) . Bars represent SEM of biological replicates .

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

A. Sanger screen IC50 drug sensitivity data (IC50 values) for DDR inhibitors targeting ATR, CHK1/2 and Wee1 in T-ALL, PTCL (DEL, KARPAS-299, SUP-M2) and pan-cancer cell lines. ANOVA p=0.413 (AZD6738, ATR inhibitor), p=0.750 (AZD772, CHK1/2 inhibitor), p=0.119 (MK-8776, ATR inhibitor) and 0.282 (AZD1775, Wee1 inhibitor). Bars represent SEM of biological replicates.


B . PTCL ( red ) and T - ALL ( yellow ) human cell lines were dosed in vitro with AZD6738 for 72h and viable cell numbers assayed by luminescence readout using Cell TiterGlo . Average IC50 values from three experiments are plotted . Bars represent SEM .

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

B. PTCL (red) and T-ALL (yellow) human cell lines were dosed in vitro with AZD6738 for 72h and viable cell numbers assayed by luminescence readout using Cell TiterGlo. Average IC50 values from three experiments are plotted. Bars represent SEM.


F . Western blot for γH2AX relative to total H2AX levels of spleen lysates from mice dosed for 71 days with AZD6738 ( ATR ; n = 4 ) relative to untreated mPTCL spleens taken at endpoint ( n = 6 ) . T - test p = 2 . 70E - 06 . PC9 cells dosed in vitro 24h with 1 µM aphidicolin ( Aph ; an inhibitor of DNA replication ) or with AZD6738 ( ATR ) are positive controls for DNA damage . Bars represent SEM . * * * p < 0 .

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

F. Western blot for γH2AX relative to total H2AX levels of spleen lysates from mice dosed for 71 days with AZD6738 (ATR; n=4) relative to untreated mPTCL spleens taken at endpoint (n=6). T-test p=2.70E-06. PC9 cells dosed in vitro 24h with 1 µM aphidicolin (Aph; an inhibitor of DNA replication) or with AZD6738 (ATR) are positive controls for DNA damage. Bars represent SEM. ***p<0.001


B ) Percentage specific binding of [ 3H ] dofetilide to the hERG channel after 6 minutes of dissociation induced by 10 µM dofetilide in the absence ( control ) or presence of 10 and 50 µM of LUF compounds . The specific binding of [ 3H ] dofetilide in the absence of test compounds was set as Bcontrol , while the specific binding in their presence was set as B . * = p < 0 . 05 vs control ; N : 3 - 4 .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O 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) Percentage specific binding of [3H]dofetilide to the hERG channel after 6 minutes of dissociation induced by 10 µM dofetilide in the absence (control) or presence of 10 and 50 µM of LUF compounds. The specific binding of [3H]dofetilide in the absence of test compounds was set as Bcontrol, while the specific binding in their presence was set as B. * = p < 0.05 vs control; N: 3-4.


C ) Representative traces of hERG activation ( left ) , inactivation ( middle ) , and deactivation ( right ) measured in HEK293 hERG cells under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . Insets : voltage clamp protocols .

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

C) Representative traces of hERG activation (left), inactivation (middle), and deactivation (right) measured in HEK293 hERG cells under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). Insets: voltage clamp protocols.


D ) Steady - state activation ( empty symbols ) and inactivation ( filled symbols ) curves for IKr under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . The corresponding Boltzmann ' s fittings are superimposed to data points . * = p < 0 . 05 vs baseline . N : 11 - 14 .

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

D) Steady-state activation (empty symbols) and inactivation (filled symbols) curves for IKr under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). The corresponding Boltzmann's fittings are superimposed to data points.* = p < 0.05 vs baseline. N: 11-14.


E ) Plot of the time constants ( τ ) of deactivation derived from biexponential fittings under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . * = p < 0 . 05 vs respective baseline . N : 8 .

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

E) Plot of the time constants (τ) of deactivation derived from biexponential fittings under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p < 0.05 vs respective baseline. N: 8.


B ) Baseline QT interval ( top ) , baseline RR interval ( middle ) , and baseline QT interval corrected with the Bazett ' s formula ( bottom ) . Bar graphs are divided by isogenic pairs ( LQT1R594Q and JLNSR594Q , left ; LQT2corr , LQT2N996I , hESCWT , and hESC - LQT2N996I , middle ; LQT1corr and LQT1R190Q , right ) and in each graph the unrelated WT is shown as a comparison . N : 17 - 53 . * = p < 0 . 05 . The colour of the symbol indicates comparisons and relative statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Baseline QT interval (top), baseline RR interval (middle), and baseline QT interval corrected with the Bazett's formula (bottom). Bar graphs are divided by isogenic pairs (LQT1R594Q and JLNSR594Q, left; LQT2corr, LQT2N996I, hESCWT, and hESC-LQT2N996I, middle; LQT1corr and LQT1R190Q, right) and in each graph the unrelated WT is shown as a comparison. N: 17-53. * = p<0.05. The colour of the symbol indicates comparisons and relative statistical significance.


C ) Representative traces of IKr steady state activation in WT hiPSC - CMs in Tyrode ( left ) , in the presence of 5 µM LUF7346 ( middle ) and after the application of 5 µM E4031 to selectively block IKr ( right ) . Inset : voltage clamp protocol .

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

C) Representative traces of IKr steady state activation in WT hiPSC-CMs in Tyrode (left), in the presence of 5 µM LUF7346 (middle) and after the application of 5 µM E4031 to selectively block IKr (right). Inset: voltage clamp protocol.


D ) Average I / V relationships ( left ) and steady - state activation curves with superimposed Boltzmann ' s fittings ( right ) under baseline conditions ( black ) and in the presence of 5 µM LUF7346 ( red ) . * = p < 0 . 05 . N : 19 .

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

D) Average I/V relationships (left) and steady-state activation curves with superimposed Boltzmann's fittings (right) under baseline conditions (black) and in the presence of 5 µM LUF7346 (red). * = p<0.05. N: 19.


E ) Plot of the time constants ( τ ) of deactivation derived from biexponential fits ( left ) and representative examples ( right ) under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . * = p < 0 . 05 . N : 17 - 21 .

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

E) Plot of the time constants (τ) of deactivation derived from biexponential fits (left) and representative examples (right) under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p<0.05. N: 17-21.


A ) Representative MEA trace ( left ) showing the effect of increasing concentrations of LUF7346 on FP contour , measured in LQT2N996I - CMs . Average data ( right ) showing the effect of LUF7346 on QT interval duration relative to baseline in CMs derived from all the hiPSC lines used in this study . * = p < 0 . 05 vs baseline . Colour in the heatmap defines the magnitude of QT shortening ( blue ) after treatment with increasing concentrations of LUF7346 . N : 7 - 14 .

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

A) Representative MEA trace (left) showing the effect of increasing concentrations of LUF7346 on FP contour, measured in LQT2N996I-CMs. Average data (right) showing the effect of LUF7346 on QT interval duration relative to baseline in CMs derived from all the hiPSC lines used in this study. * = p<0.05 vs baseline. Colour in the heatmap defines the magnitude of QT shortening (blue) after treatment with increasing concentrations of LUF7346. N: 7-14.


B ) Representative AP from LQT2corr - , LQT2N996I - , LQT1corr - , and LQT1R190Q - CMs paced at 1 Hz , under baseline conditions ( black ) and after application of increasing concentrations of LUF7346 ( colour - code is shown ) .

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

B) Representative AP from LQT2corr-, LQT2N996I-, LQT1corr-, and LQT1R190Q-CMs paced at 1 Hz, under baseline conditions (black) and after application of increasing concentrations of LUF7346 (colour-code is shown).


C ) Effect of LUF7346 on APD90 , APD70 , and APD50 , measured in LQT2corr - ( blue ) , LQT2N996I - ( green ) , LQT1corr - ( purple ) , LQT1R190Q - ( dark blue ) paced at 1 Hz ; * = p < 0 . 05 vs baseline . The colour of the asterisk indicates comparisons and respective statistical significance . ° = p < 0 . 05 vs the genetically - matched corrected control . # = p < 0 . 05 LQT1corr vs LQT2corr . N : 12 - 15 .

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

C) Effect of LUF7346 on APD90, APD70, and APD50, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), LQT1R190Q- (dark blue) paced at 1 Hz; * = p<0.05 vs baseline. The colour of the asterisk indicates comparisons and respective statistical significance. ° = p<0.05 vs the genetically-matched corrected control. # = p<0.05 LQT1corr vs LQT2corr. N: 12-15.


D ) Effect of LUF7346 on APA and Ediast , measured in LQT2corr - ( blue ) , LQT2N996I - ( green ) , LQT1corr - ( purple ) , and LQT1R190Q - CMs ( dark blue ) paced at 1 Hz . N : 12 - 15 .

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

D) Effect of LUF7346 on APA and Ediast, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), and LQT1R190Q-CMs (dark blue) paced at 1 Hz. N: 12-15.


A ) Representative MEA trace ( left ) showing the effect of 100 nM AST and increasing concentrations of LUF7346 in the presence of AST on FP contour , measured in LQT1R594Q - CMs . Average data ( right ) of the effect of AST and LUF7346 in the presence of AST on QT interval duration compared to baseline in CMs derived from all the hiPSC lines used in this study . * = p < 0 . 05 vs respective baseline . ° = p < 0 . 05 vs AST . Colour in the heatmap defines the magnitude of QT prolongation ( red ) and QT shortening ( blue ) , respectively , after treatment with AST and with increasing concentrations of LUF7346 in the presence of AST . N : 5 - 10 .

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

A) Representative MEA trace (left) showing the effect of 100 nM AST and increasing concentrations of LUF7346 in the presence of AST on FP contour, measured in LQT1R594Q-CMs. Average data (right) of the effect of AST and LUF7346 in the presence of AST on QT interval duration compared to baseline in CMs derived from all the hiPSC lines used in this study. * = p<0.05 vs respective baseline. ° = p<0.05 vs AST. Colour in the heatmap defines the magnitude of QT prolongation (red) and QT shortening (blue), respectively, after treatment with AST and with increasing concentrations of LUF7346 in the presence of AST. N: 5-10.


B ) Representative AP from WT - , LQT1R594Q - and JLNSR594Q - CMs , paced at 1 Hz , under baseline conditions ( black ) , after application of 100 nM AST ( red ) , and after addition of increasing concentrations of LUF7346 in the presence of AST ( colour - code is shown ) .

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

B) Representative AP from WT-, LQT1R594Q- and JLNSR594Q-CMs, paced at 1 Hz, under baseline conditions (black), after application of 100 nM AST (red), and after addition of increasing concentrations of LUF7346 in the presence of AST (colour-code is shown).


C ) Effect of AST and LUF7346 in the presence of AST on APD90 , APD70 , and APD50 in WT - ( grey ) , LQT1R594Q - ( red ) and JLNSR594Q - CMs ( black ) paced at 1 Hz . * = p < 0 . 05 vs respective baseline ; ° = p < 0 . 05 vs AST . N : 11 - 16 .

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

C) Effect of AST and LUF7346 in the presence of AST on APD90, APD70, and APD50 in WT- (grey), LQT1R594Q- (red) and JLNSR594Q-CMs (black) paced at 1 Hz. * = p<0.05 vs respective baseline; ° = p<0.05 vs AST. N: 11-16.


D ) Effect of AST and LUF7346 in the presence of AST on APA and Ediast in WT - ( grey ) , LQT1R594Q - ( red ) and JLNSR594Q - CMs ( black ) paced at 1 Hz . N : 11 - 16 .

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

D) Effect of AST and LUF7346 in the presence of AST on APA and Ediast in WT- (grey), LQT1R594Q- (red) and JLNSR594Q-CMs (black) paced at 1 Hz. N: 11-16.


A : Representative AP traces measured in JLNSR594Q - CMs paced at 1 Hz , under baseline conditions ( black ) and after application of either AST alone ( red ) , AST + LUF7346 3 µM ( green ) and AST + LUF7346 5 µM ( blue ) .

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

A: Representative AP traces measured in JLNSR594Q-CMs paced at 1 Hz, under baseline conditions (black) and after application of either AST alone (red), AST+LUF7346 3 µM (green) and AST+LUF7346 5 µM (blue).


B : STV values calculated on spontaneously beating hiPSC - CM clusters over 30 consecutive beats as measured with MEA . N = 7 - 24 . * = p < 0 . 05 vs respective isogenic pair .

O O O O O O O O O O O O O O O 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: STV values calculated on spontaneously beating hiPSC-CM clusters over 30 consecutive beats as measured with MEA. N = 7-24. * = p<0.05 vs respective isogenic pair.


C : STV changes ( left ) and representative examples of QT interval oscillations plotted over 30 consecutive beats ( right ) in the presence of AST and AST + LUF7346 20 µM . * = p < 0 . 05 vs Baseline . ° = p < 0 . 05 vs AST . N = 7 - 24 .

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

C: STV changes (left) and representative examples of QT interval oscillations plotted over 30 consecutive beats (right) in the presence of AST and AST+LUF7346 20 µM. * = p<0.05 vs Baseline. ° = p<0.05 vs AST. N = 7-24.


A - B . SH - SY5Y cells and primary cortical neurons were treated with 1 μM α - syn fibrils ( A ) or monomers ( B ) for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 compared to PBS , unpaired t - test .

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

A-B. SH-SY5Y cells and primary cortical neurons were treated with 1 μM α-syn fibrils (A) or monomers (B) for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01 compared to PBS, unpaired t-test.


C . Frontal cortices of A53T α - syn heterozygous transgenic mice and control mice at the age of 9 months ( old ) or 2 months ( young ) were lysed and Western blot was performed with the indicated antibodies ( n = 3 ) . * * * P < 0 . 001 compared to WT , unpaired t - test .

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

C. Frontal cortices of A53T α-syn heterozygous transgenic mice and control mice at the age of 9 months (old) or 2 months (young) were lysed and Western blot was performed with the indicated antibodies (n=3). *** P < 0.001 compared to WT, unpaired t-test.


FcγRIIB KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

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

FcγRIIB KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SHP - 1 KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

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

SHP-1 KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SHP - 2 KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

SHP-2 KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SH - SY5Y cells with 20 μM NSC87877 , 10 μM SSG and 1 μM PHPS - 1 were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

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

SH-SY5Y cells with 20 μM NSC87877, 10 μM SSG and 1 μM PHPS-1 were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h . The cells in the lower chamber were fixed and immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 against PBS , one - way ANOVA .

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

SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA.


NT , c - src KD # 1 and # 2 SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells for 12 h , and then fixed and immunostained with anti - EGFP antibody . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 compared to NT cells , one - way

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

NT, c-src KD #1 and #2 SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells, one-way ANOVA


, D . primary cortical neurons were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h . The cells in the lower chamber were fixed and immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 against PBS , one - way ANOVA .

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

, D. primary cortical neurons were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA.


, E . primary cortical neurons downregulating c - src were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells for 12 h , and then fixed and immunostained with anti - EGFP antibody . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 compared to NT cells one - way ANOVA unpaired t -

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

, E. primary cortical neurons downregulating c-src were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells one-way ANOVA unpaired t-test


SH - SY5Y cells were transfected with plasmids of WT c - src , Y529F c - src ( kinase active ) , or K297M c - src ( kinase dead ) tagged with EGFP . After 1 day , the cells were cocultured with differentiated α - syn OE SH - SY5Y cells for 12 h and then fixed and immunostained with anti - α - syn antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * P < 0 . 05 compared to mock positive cells , one - way ANOVA .

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

SH-SY5Y cells were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA.


G . FcγRIIB KD SH - SY5Y cells were transfected with plasmids of WT c - src , Y529F c - src ( kinase active ) , or K297M c - src ( kinase dead ) tagged with EGFP . After 1 day , the cells were cocultured with differentiated α - syn OE SH - SY5Y cells for 12 h and then fixed and immunostained with anti - α - syn antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * P < 0 . 05 compared to mock positive cells , one - way ANOVA .

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

G. FcγRIIB KD SH-SY5Y cells were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA.


B . A53T α - syn - EGFP and A53T α - syn - mCherry OE SH - SY5Y cells were cocultured in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 , respectively , with 50 μM RA for 5 days .

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

B. A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1, respectively, with 50 μM RA for 5 days.


C . NT or c - src KD # 1 or # 2 / A53T α - syn - EGFP and A53T α - syn - mCherry OE SH - SY5Y cells were cocultured with 50 μM RA for 5 days . The samples were observed under confocal microscopy , and the number of cells containing double fluorescence labeled puncta were analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to PBS or NT cells , one - way ANOVA .

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

C. NT or c-src KD #1 or #2 /A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured with 50 μM RA for 5 days. The samples were observed under confocal microscopy, and the number of cells containing double fluorescence labeled puncta were analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to PBS or NT cells, one-way ANOVA.


A . Differentiated A53T α - syn - EGFP OE SH - SY5Y cells were incubated with 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h .

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

A. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h.


B . Differentiated NT , c - src KD # 1 or c - src KD # 2 / A53T α - syn - EGFP OE SH - SY5Y cells were cultured 12 h . Levels of α - syn in culture media were then measured using ELISA . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 compared to control , one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Differentiated NT, c-src KD #1 or c-src KD#2/A53T α-syn-EGFP OE SH-SY5Y cells were cultured 12 h. Levels of α-syn in culture media were then measured using ELISA. Values were derived from three independent experiments (n=3). *** P < 0.001, ** P < 0.01 compared to control, one-way ANOVA.


D . SH - SY5Y cells in the lower chamber were cocultured with differentiated NT , c - src KD # 1 or c - src KD # 2 / A53T α - syn - EGFP OE SH - SY5Y cells for 12 h . The cells in the lower chamber were immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to NT cells , one - way ANOVA .

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

D. SH-SY5Y cells in the lower chamber were cocultured with differentiated NT, c-src KD #1 or c-src KD #2/ A53T α-syn-EGFP OE SH-SY5Y cells for 12 h. The cells in the lower chamber were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to NT cells, one-way ANOVA.


E . Differentiated A53T α - syn - EGFP OE SH - SY5Y cells were incubated with 5 μM saracatinib ( sara ) or 10 μM SKI - 1 in the presence of DMSO or 50 μM bafilomycin A1 ( BafA ) for 12 h .

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E. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 in the presence of DMSO or 50 μM bafilomycin A1 (BafA) for 12 h.


Differentiated NT and c - src KD / A53T α - syn - EGFP OE SH - SY5Y cells were incubated with 50 μM BafA for 12 h . Western blot was performed . Data are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one - way ANOVA .

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

Differentiated NT and c-src KD/A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.


G . NT and c - src KD primary neurons were incubated with 50 μM BafA for 12 h . Western blot was performed . Data are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one - way ANOVA .

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

G. NT and c-src KD primary neurons were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.


H . SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of DMSO or 50 μM BafA for 12 h . The cells were immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * P < 0 . 01 , one - way ANOVA .

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

H. SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of DMSO or 50 μM BafA for 12 h. The cells were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). ** P < 0.01, one-way ANOVA.


SH - SY5Y cells in the presence of 5 μM saracatinib , 10 μM SKI - 1 were incubated with 1 μM α - syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5 - LacCer and 2 . 5 μg / ml rhodamine - conjugated transferrin for 20 min . The cells were fixed and observed under confocal microscopy . The intensity was analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to control , one - way ANOVA .

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

SH-SY5Y cells in the presence of 5 μM saracatinib, 10 μM SKI-1 were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA.


B . NT , c - src KD # 1 or c - src KD # 2 SH - SY5Y cells were incubated with 1 μM α - syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5 - LacCer and 2 . 5 μg / ml rhodamine - conjugated transferrin for 20 min . The cells were fixed and observed under confocal microscopy . The intensity was analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to control , one - way ANOVA .

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

B. NT, c-src KD #1 or c-src KD #2 SH-SY5Y cells were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA.


Two to three - month - old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α - syn fibrils . The mice were treated with saracatinib at 10 mg / kg / day by oral gavage for 4 weeks . B . Fixed brain tissues containing the frontal cortex and the striatum were lysed . Western blot was performed with indicated antibodies . Values were derived from three individual mice ( n = 3 ) .

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

Two to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks. B. Fixed brain tissues containing the frontal cortex and the striatum were lysed. Western blot was performed with indicated antibodies. Values were derived from three individual mice (n=3).


Two to three - month - old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α - syn fibrils . The mice were treated with saracatinib at 10 mg / kg / day by oral gavage for 4 weeks . C . Immunohistochemistry of pSer129 α - syn was performed . Arrow indicates pSer129 α - syn positive inclusion . Scale bar indicates 50 μm . D . Scoring of pSer129 immunoreactivity in the cortex and the striatum of both regions . Values were derived from eight individual mice ( n = 8 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS group , unpaired t - test . Data are expressed as mean ± SEM .

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

Two to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks. C. Immunohistochemistry of pSer129 α-syn was performed. Arrow indicates pSer129 α-syn positive inclusion. Scale bar indicates 50 μm. D. Scoring of pSer129 immunoreactivity in the cortex and the striatum of both regions. Values were derived from eight individual mice (n=8). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS group, unpaired t-test. Data are expressed as mean ± SEM.


A . Immunohistochemistry of GFAP was performed . GFAP intensities in the cortex and the striatum were analyzed . Values were derived from three individual mice ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one - way ANOVA .

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

A. Immunohistochemistry of GFAP was performed. GFAP intensities in the cortex and the striatum were analyzed. Values were derived from three individual mice (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.


B . Immunohistochemistry of IBA - 1 and MHC Class II were performed . Arrow indicates MHC class II positive cells . Numbers of MCH class II positive cells in the cortex and the striatum were analyzed . Values were derived from three individual mice ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunohistochemistry of IBA-1 and MHC Class II were performed. Arrow indicates MHC class II positive cells. Numbers of MCH class II positive cells in the cortex and the striatum were analyzed. Values were derived from three individual mice (n=3). *** P < 0.001, ** P < 0.01, one-way ANOVA.


( A - C ) MITOL - HA formed small dot - like structures following CCCP treatment . HeLa cells transiently expressing Flag - Parkin and MITOL - HA were treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry . MITOL and Tom20 ( mitochondrial marker ; A ) signals co - localized well without CCCP treatment , whereas the MITOL - positive small dots were not coincident with Tom20 , Sec61β ( ER marker ; B ) , or LAMP1 ( lysosomal marker ; C ) after CCCP treatment . Scale bars , 10 µm . ( D ) MITOL - HA co - localized with catalase ( peroxisome marker ) following CCCP treatment . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm . Arrowheads indicate representative examples of MITOL - HA co - localization with catalase . ( E ) Correlation statistics for the localization of MITOL - HA and Tom20 , Sec61β , LAMP1 , or catalase . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test . ( F ) The line - graph shows a line scan of fluorescence through three MITOL - positive peroxisomes ( red bar in Fig 1D ) that clearly indicates co - localization of MITOL ( green line ) and catalase ( magenta line ) .

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

(A-C) MITOL-HA formed small dot-like structures following CCCP treatment. HeLa cells transiently expressing Flag-Parkin and MITOL-HA were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry. MITOL and Tom20 (mitochondrial marker; A) signals co-localized well without CCCP treatment, whereas the MITOL-positive small dots were not coincident with Tom20, Sec61β (ER marker; B), or LAMP1 (lysosomal marker; C) after CCCP treatment. Scale bars, 10 µm. (D) MITOL-HA co-localized with catalase (peroxisome marker) following CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-HA co-localization with catalase. (E) Correlation statistics for the localization of MITOL-HA and Tom20, Sec61β, LAMP1, or catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. (F) The line-graph shows a line scan of fluorescence through three MITOL-positive peroxisomes (red bar in Fig 1D) that clearly indicates co-localization of MITOL (green line) and catalase (magenta line).


( G ) Peroxisomal membrane protein ( PMP ) 34 - FusionRed also co - localized with MITOL - GFP in Parkin - expressing HeLa cells after 3 hours of CCCP treatment . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm . ( H ) Correlation statistics for the localization of MITOL - GFP and PMP34 - FusionRed . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

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

(G) Peroxisomal membrane protein (PMP) 34-FusionRed also co-localized with MITOL-GFP in Parkin-expressing HeLa cells after 3 hours of CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. (H) Correlation statistics for the localization of MITOL-GFP and PMP34-FusionRed. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( A ) MITOL - HA did not move to peroxisomes , but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin . Wild - type HeLa cells or HeLa cells stably expressing GFP - Parkin were transfected with MITOL - HA , treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - HA and anti - Tom20 antibodies . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm .

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

(A) MITOL-HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild-type HeLa cells or HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-HA and anti-Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


( B ) Correlation statistics for the localization of MITOL - HA and Tom20 in the absence or presence of GFP - Parkin . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of MITOL-HA and Tom20 in the absence or presence of GFP-Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( C ) MITOL was not degraded following mitochondrial depolarization . HeLa cells stably expressing 3Flag - MITOL were transfected with a GFP - Parkin plasmid or the pEGFP - C1 vector , treated with 15 µM CCCP + / - 10 µM MG132 for 3 hours , and then immunoblotted with anti - Flag , anti - ubiquitin , and anti - tubulin antibodies . Black arrowheads indicate 3Flag - MITOL in the upper panel and mono - ubiquitin in the middle panel , respectively .

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(C) MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag-MITOL were transfected with a GFP-Parkin plasmid or the pEGFP-C1 vector, treated with 15 µM CCCP +/- 10 µM MG132 for 3 hours, and then immunoblotted with anti-Flag, anti-ubiquitin, and anti-tubulin antibodies. Black arrowheads indicate 3Flag-MITOL in the upper panel and mono-ubiquitin in the middle panel, respectively.


( D ) The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment . Immunoblotting combined with fractionation analysis showed that Mitofusin2 ( MFN2 ) underwent rapid degradation within 3 hours of valinomycin treatment , in particular in the 3 , 000 g pellet ( mitochondria - rich fractions ) . Cytochrome c oxidase subunit 2 ( MTCO2 , inner mitochondrial protein ) was significantly reduced at 24 hours 10 μM valinomycin treatment . In contrast to those two proteins , MITOL degradation was minimal . Note that the chemical apoptosis inhibitor ZVAD - FMK ( 10 μM ) was added to cells along with valinomycin to prevent cell death .

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(D) The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that Mitofusin2 (MFN2) underwent rapid degradation within 3 hours of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria-rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 hours 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor ZVAD-FMK (10 μM) was added to cells along with valinomycin to prevent cell death.


( E ) Quantification of 3Flag - MITOL , MFN2 , and MTCO2 protein levels in the PNS and 3 , 000 g pellet fraction following 10 µM valinomycin + ZVAD - FMK treatment at the indicated times . Data represent the mean fold change ± s . e . m relative to untreated samples in three biological replicates .

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(E) Quantification of 3Flag-MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 µM valinomycin + ZVAD-FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m relative to untreated samples in three biological replicates.


( F ) Pre - existing MITOL on mitochondria moves to peroxisomes following CCCP treatment . Following doxycycline treatment for 3 hours to induce MITOL expression , cells were washed with fresh medium to stop the synthesis of new MITOL . After treatment with or without CCCP for more than 3 hours , cells were immunostained using anti - Flag , anti - Pex14 ( peroxisomal membrane protein ) , and anti - Hsp60 antibodies . Higher magnification images of the boxed regions are shown in the bottom panel . Scale bars , 10 µm .

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

(F) Pre-existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 hours to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 hours, cells were immunostained using anti-Flag, anti-Pex14 (peroxisomal membrane protein), and anti-Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 µm.


( A ) Wild - type or PINK1 knock out ( KO ) HeLa cells were transfected with GFP - Parkin wild - type or the C431S mutant , treated with 15 µM CCCP , and then immuno - stained with anti - Flag and anti - catalase antibodies . Wild - type Parkin , but not the catalytically inactive Parkin ( C431S ) mutant , mediated 3Flag - MITOL translocation to peroxisomes following mitophagy stimulation . In PINK1 KO HeLa cells , the redistribution of MITOL to peroxisomes was not observed even in the presence of wild - type Parkin when mitochondria were damaged . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm . Arrowheads indicate representative examples of MITOL - peroxisome co - localization observed only in the presence of wild - type Parkin .

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

(A) Wild-type or PINK1 knock out (KO) HeLa cells were transfected with GFP-Parkin wild-type or the C431S mutant, treated with 15 µM CCCP, and then immuno-stained with anti-Flag and anti-catalase antibodies. Wild-type Parkin, but not the catalytically inactive Parkin (C431S) mutant, mediated 3Flag-MITOL translocation to peroxisomes following mitophagy stimulation. In PINK1 KO HeLa cells, the redistribution of MITOL to peroxisomes was not observed even in the presence of wild-type Parkin when mitochondria were damaged. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-peroxisome co-localization observed only in the presence of wild-type Parkin.


( B ) The redistribution of MITOL from mitochondria to peroxisomes does not require its own E3 activity . The E3 - inactive MITOL Cys65Ser / Cys68Ser ( CS ) and H43W mutants were transfected into HeLa cells stably expressing HA - Parkin , treated with 15 µM CCCP , and then subjected to immunocytochemistry with anti - Flag and anti - catalase antibodies . After 3 hours of CCCP treatment , both the CS and H43W mutants co - localized with catalase . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm . Arrowheads indicate representative examples of 3Flag - MITOL co - localization with catalase .

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

(B) The redistribution of MITOL from mitochondria to peroxisomes does not require its own E3 activity. The E3-inactive MITOL Cys65Ser/Cys68Ser (CS) and H43W mutants were transfected into HeLa cells stably expressing HA-Parkin, treated with 15 µM CCCP, and then subjected to immunocytochemistry with anti-Flag and anti-catalase antibodies. After 3 hours of CCCP treatment, both the CS and H43W mutants co-localized with catalase. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of 3Flag-MITOL co-localization with catalase.


( C ) Correlation statistics for the localization of 3Flag - MITOL and catalase in the presence of GFP - Parkin wild - type or an inactive C431S mutant . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test . MITOL overlapped with catalase only in the presence of wild - type Parkin in wild - type HeLa cells . In PINK1 knockout ( KO ) HeLa cells , wild - type Parkin was unable to induce the peroxisomal translocation of MITOL .

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

(C) Correlation statistics for the localization of 3Flag-MITOL and catalase in the presence of GFP-Parkin wild-type or an inactive C431S mutant. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. MITOL overlapped with catalase only in the presence of wild-type Parkin in wild-type HeLa cells. In PINK1 knockout (KO) HeLa cells, wild-type Parkin was unable to induce the peroxisomal translocation of MITOL.


( D ) Correlation statistics for the localization of 3Flag - MITOL wild - type or inactive mutants with catalase . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

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

(D) Correlation statistics for the localization of 3Flag-MITOL wild-type or inactive mutants with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( B ) Peroxisomal translocation of other mitochondrial proteins such as MitoNEET / CISD1 , Fis1 , Miro1 / 2 , and Tom70 ( mitochondrial outer membrane proteins ) , and Hsp60 ( mitochondrial matrix protein ) was not observed in CCCP - treated cells . HeLa cells stably expressing HA - Parkin were treated with 15 µM CCCP for 3 hours , and then immuno - stained with anti - MitoNEET / CISD1 , anti - Fis1 , anti - Miro1 / 2 , anti - Tom70 , anti - Hsp60 , and anti - catalase antibodies . Scale bars , 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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) Peroxisomal translocation of other mitochondrial proteins such as MitoNEET/CISD1, Fis1, Miro1/2, and Tom70 (mitochondrial outer membrane proteins), and Hsp60 (mitochondrial matrix protein) was not observed in CCCP-treated cells. HeLa cells stably expressing HA-Parkin were treated with 15 µM CCCP for 3 hours, and then immuno-stained with anti-MitoNEET/CISD1, anti-Fis1, anti-Miro1/2, anti-Tom70, anti-Hsp60, and anti-catalase antibodies. Scale bars, 10 µm.


( C ) A multi - spanning outer membrane protein PBR ( Peripheral Benzodiazepine Receptor ) did not co - localize with catalase , but did with Tom20 during mitophagy . HeLa cells stably expressing HA - Parkin were treated with 15 µM CCCP for 3 hours , and then immuno - stained with anti - Flag , anti - Tom20 , and anti - catalase antibodies . Scale bars , 10 µm .

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

(C) A multi-spanning outer membrane protein PBR (Peripheral Benzodiazepine Receptor) did not co-localize with catalase, but did with Tom20 during mitophagy. HeLa cells stably expressing HA-Parkin were treated with 15 µM CCCP for 3 hours, and then immuno-stained with anti-Flag, anti-Tom20, and anti-catalase antibodies. Scale bars, 10 µm.


( A ) siRNA - based knockdown of endogenous Tom70 , Tom20 , Tom40 , and Sam50 . HeLa cells treated with the corresponding siRNAs and immunoblotted with the indicated antibodies . AIF , Apoptosis - inducing factor .

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(A) siRNA-based knockdown of endogenous Tom70, Tom20, Tom40, and Sam50. HeLa cells treated with the corresponding siRNAs and immunoblotted with the indicated antibodies. AIF, Apoptosis-inducing factor.


( B ) HeLa cells were immuno - stained with anti - Flag and anti - catalase antibodies after treatment of each siRNA . Tom20 - and Tom40 - siRNA treatment inhibited import of Su9 - GFP ( the targeting sequence of the FoF1 ATPase subunit 9 ) into the mitochondria , leading to an accumulation of Su9 - GFP precursor proteins in the cytosol and cell nucleus . 3Flag - MITOL still localized to mitochondria in Tom20 , Tom40 , and Sam50 siRNA - treated cells . In contrast , 3Flag - MITOL dispersed into the cytosol when Tom70 was knocked down . Scale bars , 10 µm .

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(B) HeLa cells were immuno-stained with anti-Flag and anti-catalase antibodies after treatment of each siRNA. Tom20- and Tom40-siRNA treatment inhibited import of Su9-GFP (the targeting sequence of the FoF1 ATPase subunit 9) into the mitochondria, leading to an accumulation of Su9-GFP precursor proteins in the cytosol and cell nucleus. 3Flag-MITOL still localized to mitochondria in Tom20, Tom40, and Sam50 siRNA-treated cells. In contrast, 3Flag-MITOL dispersed into the cytosol when Tom70 was knocked down. Scale bars, 10 µm.


( C ) Statistical analysis of the MITOL subcellular localization following 15 µM CCCP treatment for 3 hours in cells treated with control , Tom70 , Tom20 , Tom40 , or Sam50 siRNAs . Su9 - GFP was not imported into the mitochondria , but rather localized to the cytosol and nucleus following Tom20 and Tom40 knockdown . The number of HeLa cells with cytosol - localized Su9 - GFP or 3Flag - MITOL in each siRNA experiment were determined . Data represent the mean ± s . e . m from > 100 cells in three biological replicates . ( In case of Tom40 , and Sam50 siRNAs , data represent the mean ± s . e . m from > 60 cells in three biological replicates . ) Statistical significance was calculated using a one - tailed Welch ' s t - test .

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

(C) Statistical analysis of the MITOL subcellular localization following 15 µM CCCP treatment for 3 hours in cells treated with control, Tom70, Tom20, Tom40, or Sam50 siRNAs. Su9-GFP was not imported into the mitochondria, but rather localized to the cytosol and nucleus following Tom20 and Tom40 knockdown. The number of HeLa cells with cytosol-localized Su9-GFP or 3Flag-MITOL in each siRNA experiment were determined. Data represent the mean ± s.e.m from > 100 cells in three biological replicates. (In case of Tom40, and Sam50 siRNAs, data represent the mean ± s.e.m from > 60 cells in three biological replicates.) Statistical significance was calculated using a one-tailed Welch's t-test.


( D ) HeLa cells treated with siControl or siTom70 were fractionated into cytosolic and mitochondrial fractions and then immunoblotted with anti - lactate dehydrogenase ( LDH ) , anti - Tom20 , and anti - Flag antibodies . LDH was used as a cytosolic marker . Although some MITOL detached from mitochondria during fractionation , sufficient amounts of MITOL were collected in the mitochondria - enriched fraction in control cells . In contrast , almost all of the MITOL was collected in the cytosolic fraction in siTom70 - treated cells .

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

(D) HeLa cells treated with siControl or siTom70 were fractionated into cytosolic and mitochondrial fractions and then immunoblotted with anti-lactate dehydrogenase (LDH), anti-Tom20, and anti-Flag antibodies. LDH was used as a cytosolic marker. Although some MITOL detached from mitochondria during fractionation, sufficient amounts of MITOL were collected in the mitochondria-enriched fraction in control cells. In contrast, almost all of the MITOL was collected in the cytosolic fraction in siTom70-treated cells.


( A ) CRISPR / Cas9 gene editing was used to generate peroxisome - null cell lines by knocking out PEX19 . In wild - type HCT116 cells , catalase localized to peroxisomes ( dot - like structures ) , whereas in PEX19 - / - cells catalase was diffusely localized throughout the cytosol , indicating loss of peroxisomes . Scale bars , 10 µm .

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(A) CRISPR/Cas9 gene editing was used to generate peroxisome-null cell lines by knocking out PEX19. In wild-type HCT116 cells, catalase localized to peroxisomes (dot-like structures), whereas in PEX19 -/- cells catalase was diffusely localized throughout the cytosol, indicating loss of peroxisomes. Scale bars, 10 µm.


Wild - type and PEX19 - / - HCT116 cells stably expressing HA - Parkin and 3Flag - MITOL were treated with 10 µM valinomycin for 3 hours , and then subjected to immunocytochemistry with anti - Flag , anti - catalase , and anti - Hsp60 ( B ) antibodies . In wild - type HCT116 cells , MITOL translocated to peroxisomes , whereas MITOL remained associated with mitochondria in PEX19 - / - cells . Arrowheads indicate representative examples of 3Flag - MITOL co - localization with catalase rather than Hsp60 in wild - type HCT116 cells . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm .

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

Wild-type and PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were treated with 10 µM valinomycin for 3 hours, and then subjected to immunocytochemistry with anti-Flag, anti-catalase, and anti-Hsp60 (B) antibodies. In wild-type HCT116 cells, MITOL translocated to peroxisomes, whereas MITOL remained associated with mitochondria in PEX19 -/- cells. Arrowheads indicate representative examples of 3Flag-MITOL co-localization with catalase rather than Hsp60 in wild-type HCT116 cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


Wild - type and PEX19 - / - HCT116 cells stably expressing HA - Parkin and 3Flag - MITOL were treated with 10 µM valinomycin for 3 hours , and then subjected to immunocytochemistry with anti - Flag anti - Tom20 ( C ) antibodies . In wild - type HCT116 cells , MITOL translocated to peroxisomes , whereas MITOL remained associated with mitochondria in PEX19 - / - cells . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm .

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

Wild-type and PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were treated with 10 µM valinomycin for 3 hours, and then subjected to immunocytochemistry with anti-Flag anti-Tom20 (C) antibodies. In wild-type HCT116 cells, MITOL translocated to peroxisomes, whereas MITOL remained associated with mitochondria in PEX19 -/- cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


( A ) siRNA - based knockdown of Pex3 , Pex16 , and Pex19 . HeLa cell lysates treated with the indicated plasmids and siRNAs were immunoblotted with the indicated antibodies .

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(A) siRNA-based knockdown of Pex3, Pex16, and Pex19. HeLa cell lysates treated with the indicated plasmids and siRNAs were immunoblotted with the indicated antibodies.


( B ) A comparable amount of MITOL was detected in control , PEX3 - , PEX16 - , and PEX19 - knockdown cells . HeLa cell lysates stably expressing HA - Parkin and 3Flag - MITOL treated with the corresponding siRNAs were immunoblotted with the indicated antibodies .

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(B) A comparable amount of MITOL was detected in control, PEX3-, PEX16-, and PEX19-knockdown cells. HeLa cell lysates stably expressing HA-Parkin and 3Flag-MITOL treated with the corresponding siRNAs were immunoblotted with the indicated antibodies.


( C ) MITOL translocation from the mitochondria to peroxisomes is highly dependent on Pex3 . HeLa cells stably expressing 3Flag - MITOL and HA - Parkin were transfected with control , PEX3 , PEX16 , or PEX19 siRNA , treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - Flag and anti - catalase antibodies . Scale bars , 10 µm .

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(C) MITOL translocation from the mitochondria to peroxisomes is highly dependent on Pex3. HeLa cells stably expressing 3Flag-MITOL and HA-Parkin were transfected with control, PEX3, PEX16, or PEX19 siRNA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-catalase antibodies. Scale bars, 10 µm.


( D ) Correlation statistics for the localization of 3Flag - MITOL and catalase in cells transfected with the indicated siRNAs . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

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

(D) Correlation statistics for the localization of 3Flag-MITOL and catalase in cells transfected with the indicated siRNAs. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( A ) MITOL did not merge with Hsp60 in control siRNA - treated cells , whereas most MITOL co - localized with Hsp60 in p97 / VCP knockdown cells in response to mitophagy stimuli . HeLa cells stably expressing 3Flag - MITOL and HA - Parkin were transfected with control or p97 / VCP siRNA , treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - Flag , anti - catalase , and anti - Hsp60 antibodies . Scale bars , 10 µm .

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

(A) MITOL did not merge with Hsp60 in control siRNA-treated cells, whereas most MITOL co-localized with Hsp60 in p97/VCP knockdown cells in response to mitophagy stimuli. HeLa cells stably expressing 3Flag-MITOL and HA-Parkin were transfected with control or p97/VCP siRNA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag, anti-catalase, and anti-Hsp60 antibodies. Scale bars, 10 µm.


( B ) HeLa cell lysates treated with control or p97 / VCP siRNA were immunoblotted with anti - p97 / VCP and anti - tubulin antibodies .

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(B) HeLa cell lysates treated with control or p97/VCP siRNA were immunoblotted with anti-p97/VCP and anti-tubulin antibodies.