( 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 B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SUBCELLULAR B-EXP_ASSAY O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD 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 B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD 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 .

O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

(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 .

O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O

(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 B-GENEPROD O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O B-SUBCELLULAR O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O 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 B-CELL_LINE O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR 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 .

O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O

(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 B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O O B-EXP_ASSAY 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 B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O B-EXP_ASSAY 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 .

O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O O

(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 .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O

(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 .

O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O

(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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD 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 .

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O

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


( C ) Overexpression of an p97 / VCP ATP hydrolysis - defective mutant , E305Q / E578Q ( p97QQ ) , blocked MITOL redistribution from mitochondria to peroxisomes , while overexpression of wild - type p97 / VCP had no effect on MITOL redistribution . HeLa cells stably expressing GFP - Parkin were transfected with MITOL - HA and Flag - p97 / VCP wild - type or p97QQ , treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - HA and anti - catalase antibodies . 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 that was only observed in the presence of a functional VCP .

O O O O O O B-GENEPROD O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O

(C) Overexpression of an p97/VCP ATP hydrolysis-defective mutant, E305Q/E578Q (p97QQ), blocked MITOL redistribution from mitochondria to peroxisomes, while overexpression of wild-type p97/VCP had no effect on MITOL redistribution. HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA and Flag-p97/VCP wild-type or p97QQ, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-HA and anti-catalase antibodies. 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 that was only observed in the presence of a functional VCP.


( D ) NMS - 873 , a specific inhibitor of p97 / VCP , prevented MITOL translocation following CCCP - treatment . HeLa cells stably expressing HA - Parkin were transfected with 3Flag - MITOL , treated with 15 µM CCCP in the presence or absence of 10 µM NMS - 873 for 3 hours , and then subjected to immuno - staining with anti - Flag , anti - catalase , and anti - HA antibodies . 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 that was only observed in the presence of a functional VCP .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O

(D) NMS-873, a specific inhibitor of p97/VCP, prevented MITOL translocation following CCCP-treatment. HeLa cells stably expressing HA-Parkin were transfected with 3Flag-MITOL, treated with 15 µM CCCP in the presence or absence of 10 µM NMS-873 for 3 hours, and then subjected to immuno-staining with anti-Flag, anti-catalase, and anti-HA antibodies. 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 that was only observed in the presence of a functional VCP.


( E ) Correlation statistics for the localization of 3Flag - MITOL and catalase in the presence of NMS - 873 . 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of 3Flag-MITOL and catalase in the presence of NMS-873. 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 was ubiquitylated only in the presence of Parkin when the mitochondrial membrane was decreased . After 15 µM CCCP treatment for 3 hours , HeLa cells stably expressing HA - Parkin and 3Flag - MITOL were immunoprecipitated with anti - Flag magnetic beads , and then immunoblotted with the indicated antibodies . Red bars indicate ubiquitylation ; the black arrowhead indicates 3Flag - MITOL .

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(A) MITOL was ubiquitylated only in the presence of Parkin when the mitochondrial membrane was decreased. After 15 µM CCCP treatment for 3 hours, HeLa cells stably expressing HA-Parkin and 3Flag-MITOL were immunoprecipitated with anti-Flag magnetic beads, and then immunoblotted with the indicated antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates 3Flag-MITOL.


( B ) The characteristic ubiquitylation ladder was observed for wild - type MITOL , but was absent in the K268A and K54A / K268A mutants . Black arrowhead indicates 3Flag - MITOL .

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(B) The characteristic ubiquitylation ladder was observed for wild-type MITOL, but was absent in the K268A and K54A/K268A mutants. Black arrowhead indicates 3Flag-MITOL.


( C ) The MITOL K268A and K268R mutants were targeted to mitochondria under steady - state conditions , whereas peroxisomal localization following CCCP treatment was considerably disrupted . HeLa cells stably expressing HA - Parkin were transfected with 3Flag - MITOL wild - type , K268A , or K268R mutants , treated with 15 µM CCCP for 3 hours , and then subjected to immuno - staining with anti - Flag and anti - catalase antibodies . Scale bars , 10 µm .

O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O

(C) The MITOL K268A and K268R mutants were targeted to mitochondria under steady-state conditions, whereas peroxisomal localization following CCCP treatment was considerably disrupted. HeLa cells stably expressing HA-Parkin were transfected with 3Flag-MITOL wild-type, K268A, or K268R mutants, treated with 15 µM CCCP for 3 hours, and then subjected to immuno-staining with anti-Flag and anti-catalase antibodies. Scale bars, 10 µm.


( D ) The double K54 / K268 mutation did not change the MITOL localization pattern . The subcellular localization of the MITOL K268A or K268R mutants was not drastically changed by inclusion of the K54A or K54R mutations . Scale bars , 10 µm .

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(D) The double K54/K268 mutation did not change the MITOL localization pattern. The subcellular localization of the MITOL K268A or K268R mutants was not drastically changed by inclusion of the K54A or K54R mutations. Scale bars, 10 µm.


( E ) Correlation statistics for the localization of 3Flag - MITOL wild - type , K268A , or K268R 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of 3Flag-MITOL wild-type, K268A, or K268R 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.


( F ) The fold change in ubiquitylation of MITOL K268 , K40 , and K54 in valinomycin - treated samples versus untreated samples . After 3 hours of valinomycin treatment , PEX19 - / - HCT116 cells stably expressing HA - Parkin and 3Flag - MITOL were immunoprecipitated with anti - Flag magnetic beads , and then subjected to LC - MS / MS analysis for label - free quantification of ubiquitylated peptides . Error bars represent the mean ± s . e . m in three biological replicates .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(F) The fold change in ubiquitylation of MITOL K268, K40, and K54 in valinomycin-treated samples versus untreated samples. After 3 hours of valinomycin treatment, PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were immunoprecipitated with anti-Flag magnetic beads, and then subjected to LC-MS/MS analysis for label-free quantification of ubiquitylated peptides. Error bars represent the mean ± s.e.m in three biological replicates.


( A ) To generate MITOL - 3Flag knock - in ( KI ) HCT116 cell lines , 3xFlag gene cassettes were inserted upstream of the MITOL stop codon using CRISPR / Cas9 - based gene editing . Insertion of the 3Flag - tag was verified by immunoblotting with an anti - Flag antibody . Asterisk indicates a cross - reacting band .

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(A) To generate MITOL-3Flag knock-in (KI) HCT116 cell lines, 3xFlag gene cassettes were inserted upstream of the MITOL stop codon using CRISPR/Cas9-based gene editing. Insertion of the 3Flag-tag was verified by immunoblotting with an anti-Flag antibody. Asterisk indicates a cross-reacting band.


( B ) The MITOL subcellular localization was observed in MITOL - 3Flag KI HCT116 cells stably expressing HA - Parkin with anti - Flag , anti - catalase , and anti - Hsp60 antibodies . Endogenous MITOL ( detectable with an anti - Flag antibody ) overlapped with Hsp60 under steady - state conditions , whereas 3 hours of valinomycin ( 10 µM ) treatment induced translocation of endogenous MITOL from mitochondria to peroxisomes . Higher magnification images of the boxed regions are shown in the bottom panel . Scale bars , 10 µm .

O O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

(B) The MITOL subcellular localization was observed in MITOL-3Flag KI HCT116 cells stably expressing HA-Parkin with anti-Flag, anti-catalase, and anti-Hsp60 antibodies. Endogenous MITOL (detectable with an anti-Flag antibody) overlapped with Hsp60 under steady-state conditions, whereas 3 hours of valinomycin (10 µM) treatment induced translocation of endogenous MITOL from mitochondria to peroxisomes. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 µm.


( C ) Correlation statistics for the localization of endogenous MITOL - 3Flag with catalase . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines show the medians , box limits indicate the 25th and 75th percentiles as determined by the R software package , and 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 the X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 endogenous MITOL-3Flag with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and 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 the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( D ) The extraction of endogenous MITOL from depolarized mitochondria is blocked by the p97 / VCP inhibitor NMS - 873 . Higher magnification images of the boxed regions are shown in the lower panel . Scale bars , 10 µm .

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(D) The extraction of endogenous MITOL from depolarized mitochondria is blocked by the p97/VCP inhibitor NMS-873. Higher magnification images of the boxed regions are shown in the lower panel. Scale bars, 10 µm.


( E ) Correlation statistics for the localization of endogenous MITOL and PMP70 in the presence of NMS - 873 . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines show the medians , box limits indicate the 25th and 75th percentiles as determined by the R software package , and 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 the X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of endogenous MITOL and PMP70 in the presence of NMS-873. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and 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 the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( F ) Endogenous MITOL is ubiquitylated following valinomycin treatment . After MITOL - 3Flag KI HCT116 cells stably expressing HA - Parkin were treated with valinomycin for 3 hours , the collected cell lysates were immunoprecipitated with anti - Flag magnetic beads . The immunoprecipitates were blotted using anti - Flag and anti - ubiquitin antibodies . Red bars indicate ubiquitylation ; the black arrowhead indicates MITOL - 3Flag .

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(F) Endogenous MITOL is ubiquitylated following valinomycin treatment. After MITOL-3Flag KI HCT116 cells stably expressing HA-Parkin were treated with valinomycin for 3 hours, the collected cell lysates were immunoprecipitated with anti-Flag magnetic beads. The immunoprecipitates were blotted using anti-Flag and anti-ubiquitin antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates MITOL-3Flag.


( A ) Distribution of exogenous 3Flag - MITOL in the mitochondria ‐ rich or peroxisome - rich fraction following cellular fractionation . HCT116 cells stably expressing HA - Parkin and 3Flag - MITOL were subjected to fractionation and detected using anti ‐ Tom20 , anti ‐ PMP70 , and anti ‐ Flag antibodies . Tom20 and PMP70 were used as mitochondrial and peroxisomal markers , respectively . The 3 , 000 g and 100 , 000 g pellets represent the mitochondria ‐ rich and peroxisomes - rich fractions .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O

(A) Distribution of exogenous 3Flag-MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were subjected to fractionation and detected using anti‐Tom20, anti‐PMP70, and anti‐Flag antibodies. Tom20 and PMP70 were used as mitochondrial and peroxisomal markers, respectively. The 3,000 g and 100,000 g pellets represent the mitochondria‐rich and peroxisomes-rich fractions.


( B ) The recovery ratio of 3Flag - MITOL between the mitochondria - enriched ( 3 , 000 g pellet ) and peroxisome - enriched fraction ( 100 , 000 g pellet ) following valinomycin treatment for the indicated times . The ratio of peroxisome - localized 3Flag - MITOL to mitochondria - localized 3Flag - MITOL increased with valinomycin treatment for 3 hours . Graphic data represent results of two biological replicates . In scatter plot , dots indicate individual data points . Black dots indicate the ratio of 3Flag - MITOL collected in the mitochondria - enriched fractions , and red dots are the ratio of 3Flag - MITOL collected in the peroxisome - enriched fractions . Mean values are also shown .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O 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-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O

(B) The recovery ratio of 3Flag-MITOL between the mitochondria-enriched (3,000 g pellet) and peroxisome-enriched fraction (100,000 g pellet) following valinomycin treatment for the indicated times. The ratio of peroxisome-localized 3Flag-MITOL to mitochondria-localized 3Flag-MITOL increased with valinomycin treatment for 3 hours. Graphic data represent results of two biological replicates. In scatter plot, dots indicate individual data points. Black dots indicate the ratio of 3Flag-MITOL collected in the mitochondria-enriched fractions, and red dots are the ratio of 3Flag-MITOL collected in the peroxisome-enriched fractions. Mean values are also shown.


Distribution of endogenous MITOL in the mitochondria ‐ rich or peroxisome - rich fraction following cellular fractionation . The distribution of endogenous MITOL was examined using MITOL - 3Flag knock - in HCT116 cells . Treatment of cells with valinomycin for 3 hours reduced the amount of endogenous MITOL in the mitochondria - enriched fraction , but concomitantly increased endogenous MITOL in the peroxisomes - enriched fraction .

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Distribution of endogenous MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. The distribution of endogenous MITOL was examined using MITOL-3Flag knock-in HCT116 cells. Treatment of cells with valinomycin for 3 hours reduced the amount of endogenous MITOL in the mitochondria-enriched fraction, but concomitantly increased endogenous MITOL in the peroxisomes-enriched fraction.


Distribution of endogenous MITOL in the mitochondria ‐ rich or peroxisome - rich fraction following cellular fractionation . The distribution of endogenous MITOL was examined using MITOL - 3Flag knock - in HCT116 cells . Graphic data represent results of two biological replicates . In scatter plot , dots indicate individual data points . Black dots indicate the ratio of MITOL - 3Flag collected in mitochondria - enriched fractions , and red dots are the ratio of MITOL - 3Flag collected in the peroxisome - enriched fractions . Mean values are also shown .

B-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O 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-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O

Distribution of endogenous MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. The distribution of endogenous MITOL was examined using MITOL-3Flag knock-in HCT116 cells. Graphic data represent results of two biological replicates. In scatter plot, dots indicate individual data points. Black dots indicate the ratio of MITOL-3Flag collected in mitochondria-enriched fractions, and red dots are the ratio of MITOL-3Flag collected in the peroxisome-enriched fractions. Mean values are also shown.


( A ) HeLa cells expressing HA - Parkin and wild type 3Flag - MITOL or 3Flag - MITOL lacking C - terminal 8 amino acids ( ∆ C8 ) were treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - Flag and anti - PMP70 antibodies . Expanded peroxisomes were observed in MITOL ∆ C8 - expressing cells . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm .

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(A) HeLa cells expressing HA-Parkin and wild type 3Flag-MITOL or 3Flag-MITOL lacking C-terminal 8 amino acids (∆C8) were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-PMP70 antibodies. Expanded peroxisomes were observed in MITOL∆C8-expressing cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


( B ) 3Flag - MITOL ∆ C8 with the E3 - inactive Cys65Ser / Cys68Ser ( CS ) and H43W mutations were transfected into HeLa cells stably expressing HA - Parkin , treated with 15 µM CCCP , and then subjected to immunocytochemistry with anti - Flag and anti - PMP70 antibodies . After 3 hours of CCCP treatment , both the CS and H43W mutants localized on peroxisomes but expansion was not observed . Scale bars , 10 µm .

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(B) 3Flag-MITOL∆C8 with the E3-inactive Cys65Ser/Cys68Ser (CS) and H43W mutations were transfected into HeLa cells stably expressing HA-Parkin, treated with 15 µM CCCP, and then subjected to immunocytochemistry with anti-Flag and anti-PMP70 antibodies. After 3 hours of CCCP treatment, both the CS and H43W mutants localized on peroxisomes but expansion was not observed. Scale bars, 10 µm.


( C ) HeLa cells expressing HA - Parkin and MITOL wild - type or ∆ C8 were treated with 15 µM CCCP for 3 hours , and the number of peroxisomes was counted as PMP70 - positive dots per 100 μm2 . In the box - plots , dots indicate individual data points , the center lines show the medians , box limits indicate the 25th and 75th percentiles as determined by the R software package , and 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 the X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test . The abundance of peroxisomes in cells expressing MITOL ∆ C8 was significantly decreased as compared with cells expressing wild - type MITOL .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O

(C) HeLa cells expressing HA-Parkin and MITOL wild-type or ∆C8 were treated with 15 µM CCCP for 3 hours, and the number of peroxisomes was counted as PMP70-positive dots per 100 μm2. In the box-plots, dots indicate individual data points, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and 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 the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. The abundance of peroxisomes in cells expressing MITOL∆C8 was significantly decreased as compared with cells expressing wild-type MITOL.


( D ) MITOL ∆ C8 causes a drastic expansion of peroxisomes following CCCP treatment . HeLa cells expressing HA - Parkin and wild - type or ∆ C8 MITOL were treated with 15 µM CCCP for 3 hours , and the approximate size of peroxisomes was determined as the number of pixels occupied by one peroxisome . The PMP70 - positive pixels per 100 µm2 were divided by the number of peroxisomes in the same area . In the box - plots , dots indicate individual data points , the center lines show the medians , box limits indicate the 25th and 75th percentiles as determined by the R software package , and 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 the X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) MITOL∆C8 causes a drastic expansion of peroxisomes following CCCP treatment. HeLa cells expressing HA-Parkin and wild-type or ∆C8 MITOL were treated with 15 µM CCCP for 3 hours, and the approximate size of peroxisomes was determined as the number of pixels occupied by one peroxisome. The PMP70-positive pixels per 100 µm2 were divided by the number of peroxisomes in the same area. In the box-plots, dots indicate individual data points, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and 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 the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( A and B ) HeLa cells expressing HA - Parkin and MITOL ∆ C8 ( A ) or MITOL ∆ C8 lacking E3 activity ( B ) were treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - Flag and anti - ubiquitin antibodies . Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

(A and B) HeLa cells expressing HA-Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


( A ) Representative 5 - day - old seedlings of wild - type Arabidopsis thaliana Col - 0 and TF mutants gata12 and wri3 grown in the dark . Scale bar , 1 mm .

O O O O O O O O O B-ORGANISM O O O O B-ORGANISM I-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

(A) Representative 5-day-old seedlings of wild-type Arabidopsis thaliana Col-0 and TF mutants gata12 and wri3 grown in the dark. Scale bar, 1 mm.


( B ) Heat map indicates which TFs significantly affects hypocotyl length , root length and the composite trait of root to total length in dark - grown seedlings and whether the effects of TFs are dependent on condition . TFs are hierarchically clustered using Euclidean distance .

O O O O O O O O O O B-TISSUE O O B-TISSUE O O O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

(B) Heat map indicates which TFs significantly affects hypocotyl length, root length and the composite trait of root to total length in dark-grown seedlings and whether the effects of TFs are dependent on condition. TFs are hierarchically clustered using Euclidean distance.


( C ) Heat map of the average relative effects of TF mutant alleles on hypocotyl length , root length and the ratio of root to total length reveal that TF lesions significantly perturbed TCA cycle - dependent growth . Mutant alleles are listed in rows and traits in columns . Cells of TF mutant alleles in heat map are colored if the AGI or AGI : TCA Metabolite linear model terms for each trait are statistically significant ( P < 0 . 05 , two - way ANOVA , 16 - 20 seedlings per genotype per condition across two experiments per genotype ) . Mutant alleles are hierarchically clustered using Euclidean distance .

O O O O O O O O O O O O O O O B-TISSUE O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O

(C) Heat map of the average relative effects of TF mutant alleles on hypocotyl length, root length and the ratio of root to total length reveal that TF lesions significantly perturbed TCA cycle-dependent growth. Mutant alleles are listed in rows and traits in columns. Cells of TF mutant alleles in heat map are colored if the AGI or AGI:TCA Metabolite linear model terms for each trait are statistically significant (P < 0.05, two-way ANOVA, 16-20 seedlings per genotype per condition across two experiments per genotype). Mutant alleles are hierarchically clustered using Euclidean distance.


( A ) Heat map indicates which TFs significantly affects rosette area , growth rate , shoot biomass , flowering time and seed yield and whether the effects of TFs are dependent on salt treatment . TFs are hierarchically clustered using Euclidean distance .

O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(A) Heat map indicates which TFs significantly affects rosette area, growth rate, shoot biomass, flowering time and seed yield and whether the effects of TFs are dependent on salt treatment. TFs are hierarchically clustered using Euclidean distance.


( C ) Rosette area of mutant allele of BPC4 from day 7 to day 21 post - germination . Linear model for two - way ANOVA considers AGI , salt treatment , day post - germination and their interactions . The AGI and AGI : Salt Condition terms are statistically signification ( P < 0 . 001 , P < 0 . 01 , respectively ) . Heat map under line plot indicates which term in linear model is statistically significant ( P < 0 . 05 ) using two - way ANOVA for each day from 9 biological replicates per condition . Solid lines , Col - 0 ; dashed line , bpc4 ; circle , control condition ; triangle , 50 mM NaCl .

O O O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O

(C) Rosette area of mutant allele of BPC4 from day 7 to day 21 post-germination. Linear model for two-way ANOVA considers AGI, salt treatment, day post-germination and their interactions. The AGI and AGI:Salt Condition terms are statistically signification (P < 0.001, P < 0.01, respectively). Heat map under line plot indicates which term in linear model is statistically significant (P < 0.05) using two-way ANOVA for each day from 9 biological replicates per condition. Solid lines, Col-0; dashed line, bpc4; circle, control condition; triangle, 50 mM NaCl.


( A ) Hybridization Chain Reaction ( HCR ) detection of CGG repeat RNA along with immunocytochemistry ( ICC ) to detect SRSF1 - FLAG in U2OS cells . Expanded view of merged channels shows the CGG RNA foci ( arrowhead ) and an example of SRSF1 - CGG RNA foci co - localization ( arrow ) . DAPI marks the nucleus . Scale bars are 10 µm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O

(A) Hybridization Chain Reaction (HCR) detection of CGG repeat RNA along with immunocytochemistry (ICC) to detect SRSF1-FLAG in U2OS cells. Expanded view of merged channels shows the CGG RNA foci (arrowhead) and an example of SRSF1-CGG RNA foci co-localization (arrow). DAPI marks the nucleus. Scale bars are 10 µm.


( B ) Co - localization analysis of CGG RNA ( red traces ) and SRSF1 ( green traces ) showing overlaps of CGG RNA and SRSF1 . ( a . u . = arbitrary unit )

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O

(B) Co-localization analysis of CGG RNA (red traces) and SRSF1 (green traces) showing overlaps of CGG RNA and SRSF1. (a.u. = arbitrary unit)


( C ) Co - immunoprecipitation of indicated SRSF proteins with PP7 - tagged control sequence ( partial nLUC sequence ) to correct for non - specific binding , AUG and CGG reporter RNAs . SRSF1 and 2 specifically immunoprecipitated with PP7 - tagged CGG reporter .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O

(C) Co-immunoprecipitation of indicated SRSF proteins with PP7-tagged control sequence (partial nLUC sequence) to correct for non-specific binding, AUG and CGG reporter RNAs. SRSF1 and 2 specifically immunoprecipitated with PP7-tagged CGG reporter.


( D ) Representative photographs of fly eyes and quantitation ( below ) of rough eye scores with fly SRSF1 overexpression ( dSF2 OE ) ; n = 20 - 32 / genotype . Error bars represent mean + / - SD . Data information : For eye scoring , target siRNA lines were compared to non - targeting control siRNA lines using a two - tailed student ' s t test with Welch ' s correction for multiple comparisons . ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 . Human orthologs of fly genes are used for labeling .

O O O O O O B-ORGANISM B-TISSUE O O O O O O O B-TISSUE O O B-ORGANISM B-GENEPROD O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O B-ORGANISM O O O O O O

(D) Representative photographs of fly eyes and quantitation (below) of rough eye scores with fly SRSF1 overexpression (dSF2 OE); n=20-32/genotype. Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling.


( F ) Quantitation of ( GGGGCC ) 28 - EGFP rough eye phenotype with SRSF modifiers ( n > 30 flies / genotype ) . Error bars represent mean + / - SD . Data information : For eye scoring , target siRNA lines were compared to non - targeting control siRNA lines using a two - tailed student ' s t test with Welch ' s correction for multiple comparisons . ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 . Human orthologs of fly genes are used for labeling .

O O O O O O O O O O B-GENEPROD O B-TISSUE O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O B-ORGANISM O O O O O O

(F) Quantitation of (GGGGCC)28-EGFP rough eye phenotype with SRSF modifiers (n > 30 flies/ genotype). Error bars represent mean +/- SD. Data information: For eye scoring, target siRNA lines were compared to non-targeting control siRNA lines using a two-tailed student's t test with Welch's correction for multiple comparisons. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Human orthologs of fly genes are used for labeling.


( G ) Representative photographs of fly eyes expressing GMR - GAL4 driven ( GGGGCC ) 28 - EGFP at 29 ˚ C along with the quantifications of necrosis and eye width . n = 28 - 30 / genotype . Error bars represent mean + / - SD .

O O O O O O B-ORGANISM B-TISSUE O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-DISEASE O B-TISSUE O O O O O O O O O O O O O O O O O O O

(G) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP at 29˚C along with the quantifications of necrosis and eye width. n=28-30/genotype. Error bars represent mean +/- SD.


( H - I ) Survival assays of flies expressing ( CGG ) 90 - EGFP under Tub5 - GS ( H ) and ELAV - GS ( I ) drivers with control or SRSF1 siRNAs . Expression of ( CGG ) 90 - EGFP was initiated with addition of drug starting 1 day post eclosion and continued through experiment ( Log - rank Mantel - Cox test ; n = 98 - 101 / genotype for Tub - GS and n = 120 - 141 / genotype for ELAV - GS flies ) ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 . ( J ) Survival assays of ( GGGGCC ) 28 - EGFP expressing fly under Tub5 - GS driver ( Log - rank Mantel - Cox test ; n = 71 - 93 / genotype ) with control or SRSF1 siRNAs . ∗ ∗ p < 0 . 01 .

O O O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O 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-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(H-I) Survival assays of flies expressing (CGG)90-EGFP under Tub5-GS (H) and ELAV-GS (I) drivers with control or SRSF1 siRNAs. Expression of (CGG)90-EGFP was initiated with addition of drug starting 1 day post eclosion and continued through experiment (Log-rank Mantel-Cox test; n=98-101/genotype for Tub-GS and n=120-141/genotype for ELAV-GS flies) ∗p < 0.05, ∗∗p < 0.01. (J) Survival assays of (GGGGCC)28-EGFP expressing fly under Tub5-GS driver (Log-rank Mantel-Cox test; n=71-93/genotype) with control or SRSF1 siRNAs. ∗∗p < 0.01.


( A , B ) Representative ( A ) photographs of fly eyes and ( B ) quantitation with siRNA mediated knockdown of SRPK1 ( dSRPK1 ) ; n = 30 - 34 / genotype . Error bars represent mean + / - SD . Data information : Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative (A) photographs of fly eyes and (B) quantitation with siRNA mediated knockdown of SRPK1 (dSRPK1); n=30-34/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


( C ) Representative photographs of fly eyes expressing GMR - GAL4 driven ( GGGGCC ) 28 - EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion . ( D ) Quantitation of rough eye phenotypes . t - test with Welch corrections for comparisons with the control ; n = 31 - 34 flies / genotype . Error bars represent mean + / - SD . Data information : Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O O O O B-ORGANISM B-TISSUE O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (D) Quantitation of rough eye phenotypes. t-test with Welch corrections for comparisons with the control; n= 31-34 flies/ genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


( E ) Representative external eye imaging for detection of GFP aggregates caused by ( CGG ) 90 - EGFP transgene expression ( top ) . Converted images used to quantify total intensity of GFP puncta ( bottom ) . ( F ) Depletion of SRSF1 or SRPK1 by RNAi results in reduced ( CGG ) 90 - EGFP puncta compared to control siRNA as quantified by total intensity ( a . u . = arbitrary unit ) . n = 13 - 15 flies / genotype . Error bars represent mean + / - SD . Data information : Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O O O B-TISSUE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Representative external eye imaging for detection of GFP aggregates caused by (CGG)90-EGFP transgene expression (top). Converted images used to quantify total intensity of GFP puncta (bottom). (F) Depletion of SRSF1 or SRPK1 by RNAi results in reduced (CGG)90-EGFP puncta compared to control siRNA as quantified by total intensity (a.u. = arbitrary unit). n=13-15 flies/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


( B ) Anti - FLAG immunoblot of DMSO and SRPIN340 pre - treated HEK293T cells expressing AUG - nLuc - 3xFLAG control or CGG - nLuc - 3xFLAG RAN translation reporters . β - Actin is used as a loading control . To prevent signal saturation , AUG - nLuc lysate was diluted 1 : 3 in sample buffer prior to loading ( n = 3 biological replicates ) . Schematics of the AUG - nLUC - 3xFLAG and + 1CGG ( 100 ) - nLuc - 3xFLAG reporters presented on top . Data information : Error bars represent mean + / - SD . Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 . To prevent over - exposure , the AUG - nLuc lysate was diluted 1 : 3 in sample buffer .

O O O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3 biological replicates). Schematics of the AUG-nLUC-3xFLAG and +1CGG(100)-nLuc-3xFLAG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in sample buffer.


( D ) Anti - FLAG immunoblot of DMSO and SRPIN340 pre - treated HEK293T cells expressing GGGGCC - nLuc - 3xFLAG ( GA70 ) and + 2CGG - nLuc - 3xFLAG ( FMRpolyA ) RAN translation reporters ( n = 3 biological replicates ) . Schematics of the GA70 ( GGGGCCx70 ) and + 2CGG reporters presented on top . Data information : Error bars represent mean + / - SD . Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O O O B-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( E ) Immunoblot of DMSO and SPHINX31 pre - treated HEK293T cells expressing AUG - nLuc - 3xFLAG control or CGG - nLuc - 3xFLAG RAN translation reporters . ( n = 3 biological replicates ) . ( F ) Anti - FLAG of DMSO and SPHINX31 pre - treated HEK293T cells expressing GGGGCC - nLuc - 3xFLAG ( GA70 ) and + 2CGG - nLuc - 3xFLAG ( FMRpolyA ) RAN translation reporters ( n = 3 biological replicates ) . Data information : Error bars represent mean + / - SD . Statistical analysis was performed using two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O 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-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3 biological replicates). (F) Anti-FLAG of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( C , D ) Expression of GGGGCC - nLuc - 3xFLAG ( C ) and + 2CGG - nLuc - 3xFLAG ( D ) RAN translation reporters in HEK293T cells treated with 2 μM TG ( for stress induction ) analyzed by immunoblot ( n = 3 biological replicates ) . To evaluate effects of SRPK1 inhibition cells were pre - treated with DMSO or SRPIN340 before reporter transfection . Data information : Error bars represent mean + / - SD . Two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Expression of GGGGCC-nLuc-3xFLAG (C) and +2CGG-nLuc-3xFLAG (D) RAN translation reporters in HEK293T cells treated with 2 μM TG (for stress induction) analyzed by immunoblot (n=3 biological replicates). To evaluate effects of SRPK1 inhibition cells were pre-treated with DMSO or SRPIN340 before reporter transfection. Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( E ) SRPK1 inhibition by SRPIN340 did not alter eIF2α phosphorylation in response to 2 μM TG ( for stress induction ) in HEK293T cells by immunoblot ( n = 3 biological replicates ) . ( F ) SPRK1 inhibition suppressed eIF2α phosphorylation in HEK293T cells treated with 2 μM TG ( for stress induction ) in the presence of the + 1CGG reporter . Cells were pre - treated with DMSO or SRPIN340 before reporter transfection ( n = 3 biological replicates ) . Data information : Error bars represent mean + / - SD . Two - tailed Student ' s t test with Welch ' s correction . ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 and ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SRPK1 inhibition by SRPIN340 did not alter eIF2α phosphorylation in response to 2 μM TG (for stress induction) in HEK293T cells by immunoblot (n=3 biological replicates). (F) SPRK1 inhibition suppressed eIF2α phosphorylation in HEK293T cells treated with 2 μM TG (for stress induction) in the presence of the +1CGG reporter. Cells were pre-treated with DMSO or SRPIN340 before reporter transfection (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


( B ) ICC images of FLAG - tagged SRSF1 after SRPIN340 treatment compared to vehicle ( DMSO ) . Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal Error bars indicate mean + / - 95 % CI ( n = 85 - 126 cells / condition ) . Statistical analysis was performed using t test with Welch ' s correction , ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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) ICC images of FLAG-tagged SRSF1 after SRPIN340 treatment compared to vehicle (DMSO). Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal Error bars indicate mean +/- 95% CI (n= 85-126 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.


( C ) Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle ( DMSO ) as detected by HCR . Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal as parts of whole . Error bars indicate mean + / - 95 % CI ( n = 124 - 151 cells / condition ) . Statistical analysis was performed using t test with Welch ' s correction , ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle (DMSO) as detected by HCR. Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal as parts of whole. Error bars indicate mean +/- 95% CI (n= 124-151 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.


( D ) Anti - FLAG immunoblot blot of DMSO and SRPIN340 pre - treated HEK293T cells transfected with in vitro transcribed CGG - nLuc - 3xFLAG reporter RNA . β - Actin is used as a loading control . Error bars represent mean + / - SD ( n = 6 biological replicates ) . Statistical analysis was performed using Student ' s t test with Welch ' s correction . ∗ p < 0 .

O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O 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) Anti-FLAG immunoblot blot of DMSO and SRPIN340 pre-treated HEK293T cells transfected with in vitro transcribed CGG-nLuc-3xFLAG reporter RNA. β-Actin is used as a loading control. Error bars represent mean +/- SD (n=6 biological replicates). Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05


( A , B ) Pharmacological targeting of SPRK1 with ( A ) 40 μM SRPIN340 or ( B ) 8 μM SPHINX31 reduced the cumulative risk of death in + 1 ( CGG ) 100 - EGFP ( encoding FMRpolyG ) expressing neurons . n = # of neurons quantified for each condition ; Cox proportional hazard analysis , ∗ ∗ ∗ p < 0 . 001 .

O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(A, B) Pharmacological targeting of SPRK1 with (A) 40 μM SRPIN340 or (B) 8 μM SPHINX31 reduced the cumulative risk of death in +1(CGG)100-EGFP (encoding FMRpolyG) expressing neurons. n= # of neurons quantified for each condition; Cox proportional hazard analysis, ∗∗∗p < 0.001.


C Quantitative analysis of lysosomal movement from kymographs . Results were expressed as percent of total lysosomes ( N = 44 , 49 , and 16 neurons for shSc , shLAMTOR1 , and shLAMTOR + rLAMTOR1 respectively from 3 - 10 independent experiments ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by two - way ANOVA with Tukey ' s * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as compared to shSc ; # P < 0 . 05 , # # # P < 0 . 001 , as compared to shLAMTOR1 n . s . , not significant .

O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantitative analysis of lysosomal movement from kymographs. Results were expressed as percent of total lysosomes (N = 44, 49, and 16 neurons for shSc, shLAMTOR1, and shLAMTOR+rLAMTOR1 respectively from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's *P < 0.05, **P < 0.01, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1 n.s., not significant.


G FRAP analysis of dendritic lysosome movement . Quantitative analysis for F ( N = 7 , 7 , and 9 neurons for shSc , shLAMTOR1 , and shLAMTOR + rLAMTOR1 , respectively , from 3 independent experiments ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by Dunnett ' s post - test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as compared to shSc ; # P < 0 . 05 , # # # P < 0 . 001 , as compared to shLAMTOR1 ; n . s . , not significant .

O B-EXP_ASSAY O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FRAP analysis of dendritic lysosome movement. Quantitative analysis for F (N = 7, 7, and 9 neurons for shSc, shLAMTOR1, and shLAMTOR+rLAMTOR1, respectively, from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Dunnett's post-test *P < 0.05, **P < 0.01, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1 ; n.s., not significant.


A Representative kymographs of LysoTracker - labeled lysosomes in proximal dendrites of hippocampal neurons infected with scrambled shRNA ( shSc ) , LAMTOR2 shRNA ( shLAMTOR2 ) , or Raptor shRNA ( shRaptor ) . Scale bar , 5 µm . B Quantitative analysis of lysosomal movement from kymographs ( N = 44 , 24 , and 19 neurons for shSc , shLAMTOR2 , and shRaptor , respectively , from 3 - 10 independent experiments ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by two - way ANOVA with Sidak ' s post - test * * * P < 0 . 001 as compared to shSc ; n . s . , not significant .

O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative kymographs of LysoTracker-labeled lysosomes in proximal dendrites of hippocampal neurons infected with scrambled shRNA (shSc), LAMTOR2 shRNA (shLAMTOR2), or Raptor shRNA (shRaptor). Scale bar, 5 µm. B Quantitative analysis of lysosomal movement from kymographs (N = 44, 24, and 19 neurons for shSc, shLAMTOR2, and shRaptor, respectively, from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Sidak's post-test ***P < 0.001 as compared to shSc; n.s., not significant.


C , D FRAP analysis of dendritic lysosomal movement in Raptor KD neurons . C Representative images of a neuron at 0 and 4 min after photobleaching . D Quantification of the lysosomes in C undergoing retrograde ( red ) or anterograde ( cyan ) transport ( N = 7 and 8 neurons for shSc and shRaptor , respectively , from 3 independent experiments ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by Mann - Whitney U test * * * P < 0 . 001 as compared to shSc ; n . s . , not significant .

O O O B-EXP_ASSAY O O B-CELL_TYPE B-SUBCELLULAR O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D FRAP analysis of dendritic lysosomal movement in Raptor KD neurons. C Representative images of a neuron at 0 and 4 min after photobleaching. D Quantification of the lysosomes in C undergoing retrograde (red) or anterograde (cyan) transport (N = 7 and 8 neurons for shSc and shRaptor, respectively, from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant.


E LAMTOR1 KD did not affect trafficking of vesicles labeled with Alexa 594 - conjugated transferrin ( red , Tf 594 ) in dendrites . Shown are dendritic segments ( upper ) and kymographs ( lower ) . Scale bar , 5 µm . F Quantitative analysis of vesicular movement from kymographs ( N = 10 neurons from 3 independent experiments ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by Mann - Whitney U test * * * P < 0 . 001 as compared to shSc ; n . s . , not significant .

O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 LAMTOR1 KD did not affect trafficking of vesicles labeled with Alexa 594-conjugated transferrin (red, Tf 594) in dendrites. Shown are dendritic segments (upper) and kymographs (lower). Scale bar, 5 µm. F Quantitative analysis of vesicular movement from kymographs (N = 10 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant.


A Quantification of the percent of lysosomes moving in the anterograde or retrograde direction in neurons infected with AAV expressing either LAMTOR1 shRNA ( shLAMTOR1 ) or scrambled shRNA ( shSc ) and Accell lyspersin siRNA or control siRNA . Neurons were imaged with LysoTracker to visualize lysosomal trafficking in dendrites . N = 17 - 31 neurons from 3 - 6 independent experiments . Data information : Data with error bars are represented as means ± SEM . * * * P < 0 . 001 compared with shSc or shSc / Accell siControl ; # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 or shLAMTOR1 / Accell siControl ; & & P < 0 . 01 , & & & P < 0 . 001 compared with shSc / Accell siLyspersin ; n . s . , not significant ; two - way ANOVA with Tukey ' s post - test .

O O O O O O B-SUBCELLULAR O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of the percent of lysosomes moving in the anterograde or retrograde direction in neurons infected with AAV expressing either LAMTOR1 shRNA (shLAMTOR1) or scrambled shRNA (shSc) and Accell lyspersin siRNA or control siRNA. Neurons were imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17-31 neurons from 3-6 independent experiments. Data information: Data with error bars are represented as means ± SEM. ***P < 0.001 compared with shSc or shSc/Accell siControl; ##P < 0.01, ###P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; &&P < 0.01, &&&P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two-way ANOVA with Tukey's post-test.


C - F Quantification of the percent of lysosomes moving in the anterograde or retrograde direction . C , E , F Cultured hippocampal neurons were infected with AAV expressing either shLAMTOR1 or shSc ; they were treated with vehicle control or ML - SI1 ( 20 µM , C , n = 16 - 49 neurons from 3 - 10 independent experiments ) , or ML - SA1 ( 20 µM , E , n = 13 - 49 neurons from 3 - 10 independent experiments ) , or CilioD ( 20 µM , F , n = 12 - 49 neurons from 3 - 10 independent experiments ) , and imaged with LysoTracker to visualize lysosomal trafficking in dendrites . D Neurons were infected with shLAMTOR1 or shSc AAV and Accell TRPML1 siRNA or control siRNA ; they were imaged as described above . N = 7 - 31 neurons from 3 - 6 independent experiments . Data information : Data with error bars are represented as means ± SEM . * * * P < 0 . 001 compared with shSc or shSc / Accell siControl ; # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 or shLAMTOR1 / Accell siControl ; & & P < 0 . 01 , & & & P < 0 . 001 compared with shSc / Accell siLyspersin ; n . s . , not significant ; two - way ANOVA with Tukey ' s post - test .

O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Quantification of the percent of lysosomes moving in the anterograde or retrograde direction. C, E, F Cultured hippocampal neurons were infected with AAV expressing either shLAMTOR1 or shSc; they were treated with vehicle control or ML-SI1 (20 µM, C, n = 16-49 neurons from 3-10 independent experiments), or ML-SA1 (20 µM, E, n = 13-49 neurons from 3-10 independent experiments), or CilioD (20 µM, F, n = 12-49 neurons from 3-10 independent experiments), and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. D Neurons were infected with shLAMTOR1 or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were imaged as described above. N = 7-31 neurons from 3-6 independent experiments. Data information: Data with error bars are represented as means ± SEM. ***P < 0.001 compared with shSc or shSc/Accell siControl; ##P < 0.01, ###P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; &&P < 0.01, &&&P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two-way ANOVA with Tukey's post-test.


F Quantification of co - IP results . N = 3 independent experiments . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by one - way ANOVA with Dunnett ' s post - test ( F ) . * P < 0 . 05 , * * * P < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Quantification of co-IP results. N = 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test (F). *P < 0.05, ***P < 0.001.


A Interactions between LAMTOR1 and TRPML1 in mouse hippocampus . Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT - 2031 peptide . Wes protein analysis with anti - LAMTOR1 and - TRPML1 antibodies of immunoprecipitation performed with anti - LAMTOR1 antibodies or negative control anti - HA antibodies using whole hippocampal homogenates from naïve , TAT or TAT - 2031 - treated mice . B Quantification of the relative abundance of TPRML1 pulled down by LAMTOR1 in naïve , TAT or TAT - 2031 - treated mice . N = 3 mice for each group . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by one - way ANOVA with Dunnett ' s post - test * * P < 0 . 01 , * * * P < 0 . 001 .

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-ORGANISM B-TISSUE O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-TISSUE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Interactions between LAMTOR1 and TRPML1 in mouse hippocampus. Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT-2031 peptide. Wes protein analysis with anti-LAMTOR1 and -TRPML1 antibodies of immunoprecipitation performed with anti-LAMTOR1 antibodies or negative control anti-HA antibodies using whole hippocampal homogenates from naïve, TAT or TAT-2031-treated mice. B Quantification of the relative abundance of TPRML1 pulled down by LAMTOR1 in naïve, TAT or TAT-2031-treated mice. N = 3 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001.


C Representative images from proximity ligation assay ( PLA ) performed on brain slices from naïve , TAT or TAT - 2031 - treated mice . Evidence of proximity between LAMTOR1 and TRPML1 is indicated by the appearance of red puncta . Nuclei are counterstained with DAPI ( blue ) . Scale bar , 10 μm . D Quantification of the number of PLA signals in CA1 from naïve , TAT or TAT - 2031 - treated mice . N = 6 mice for each group . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by one - way ANOVA with Dunnett ' s post - test * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-TISSUE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-TISSUE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images from proximity ligation assay (PLA) performed on brain slices from naïve, TAT or TAT-2031-treated mice. Evidence of proximity between LAMTOR1 and TRPML1 is indicated by the appearance of red puncta. Nuclei are counterstained with DAPI (blue). Scale bar, 10 μm. D Quantification of the number of PLA signals in CA1 from naïve, TAT or TAT-2031-treated mice. N = 6 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001.


D TAT - 2031 treatment ( 10 µM ) increased ML - SA1 - induced TRPML1 Ca2 + release in both control and LAMTOR1 KD cells . Data information : traces represent the mean values of each group .

O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O

D TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1 Ca2+ release in both control and LAMTOR1 KD cells. Data information: traces represent the mean values of each group.


G Treatment with GPN ( 200 µM ) , BAPTA - AM ( 20 µM ) , or ML - SI1 ( 20 µM ) blocked ML - SA1 - induced TRPML1 - GCaMP6m responses in neurons . Data information : traces represent the mean values of each group .

O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O

G Treatment with GPN (200 µM), BAPTA-AM (20 µM), or ML-SI1 (20 µM) blocked ML-SA1-induced TRPML1-GCaMP6m responses in neurons. Data information: traces represent the mean values of each group.