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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O O O O O O O B-ORGANISM O O O B-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-TISSUE O O B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O

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


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

O O O O O O O O O B-ORGANISM O O O B-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-TISSUE I-TISSUE 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

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


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

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE I-TISSUE 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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O O O B-CELL_LINE O O O O B-GENEPROD 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 localization of 3Flag - MITOL wild - type or inactive mutants with catalase . Dots indicate Pearson correlation coefficient data points . In box - plots , center lines indicate medians , box limits indicate 25th 75th percentiles as determined in R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - .

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

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

O O O B-SUBCELLULAR B-EXP_ASSAY O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE 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 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 PBR ( Peripheral Benzodiazepine Receptor ) did not co - localize with catalase , but did with Tom20 during mitophagy . HeLa stably expressing HA - Parkin were treated with 15 µM CCCP for 3 hours , then immuno - stained with anti - Flag , anti - Tom20 , anti - catalase antibodies . Scale bars , 10 µm .

O O O O O O O B-SUBCELLULAR 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 B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE 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 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 - knockdown of endogenous Tom70 , Tom20 , Tom40 , Sam50 . HeLa treated with corresponding siRNAs immunoblotted with indicated antibodies . AIF , Apoptosis - inducing factor .

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

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

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

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

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

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


( A ) CRISPR / Cas9 gene editing was used to generate peroxisome - null lines by knocking out PEX19 . In wild - type HCT116 , catalase localized to peroxisomes ( dot - like structures ) , whereas in PEX19 - / - catalase was diffusely localized throughout 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 B-GENEPROD O O O O O B-CELL_LINE O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY 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 PEX19 - / - HCT116 stably expressing HA - Parkin 3Flag - MITOL were treated with 10 µM valinomycin for 3 hours , then subjected to immunocytochemistry with anti - Flag , anti - catalase , anti - Hsp60 ( B ) antibodies . In wild - type HCT116 , MITOL translocated to peroxisomes , whereas MITOL remained associated with mitochondria in PEX19 - / - . Arrowheads indicate representative examples of 3Flag - MITOL co - localization with catalase rather than Hsp60 in wild - type HCT116 . Higher magnification of boxed regions are shown in small panel . Scale bars , 10 µm .

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

O O O B-GENEPROD O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE 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 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 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 - knockdown of Pex3 , Pex16 , Pex19 . HeLa lysates treated with indicated plasmids siRNAs were immunoblotted with indicated antibodies .

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


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

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY 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 mitochondria to peroxisomes is highly dependent on Pex3 . HeLa stably expressing 3Flag - MITOL HA - Parkin were transfected with control , PEX3 , PEX16 , or PEX19 siRNA , treated with 15 µM CCCP for 3 hours , then subjected to immunocytochemistry with anti - Flag anti - catalase antibodies . Scale bars , 10 µm .

O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-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 B-EXP_ASSAY O O O B-GENEPROD 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 localization of 3Flag - MITOL catalase in transfected with indicated siRNAs . Dots indicate Pearson correlation coefficient data points . In box - plots , center lines indicate medians , box limits indicate 25th 75th percentiles as determined in R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - .

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

(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 , whereas most MITOL co - localized with Hsp60 in p97 / VCP knockdown in response to mitophagy stimuli . HeLa stably expressing 3Flag - MITOL HA - Parkin were transfected with control or p97 / VCP siRNA , treated with 15 µM CCCP for 3 hours , then subjected to immunocytochemistry with anti - Flag , anti - catalase , 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 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 B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-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 B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD 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 lysates treated with control or p97 / VCP siRNA were immunoblotted with anti - p97 / VCP anti - tubulin antibodies .

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


( 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 stably expressing GFP - Parkin were transfected with MITOL - HA Flag - p97 / VCP wild - type or p97QQ , treated with 15 µM CCCP for 3 hours , then subjected to immunocytochemistry with anti - HA anti - catalase antibodies . Higher magnification of boxed regions are shown in small panel . Scale bars , 10 µm . Arrowheads indicate representative examples of MITOL - peroxisome co - localization that was only observed in 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 B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-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 B-EXP_ASSAY 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 B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 stably expressing HA - Parkin were transfected with 3Flag - MITOL , treated with 15 µM CCCP in presence or absence of 10 µM NMS - 873 for 3 hours , then subjected to immuno - with anti - Flag , anti - catalase , anti - HA antibodies . Higher magnification of boxed regions are shown in small panel . Scale bars , 10 µm . Arrowheads indicate representative examples of MITOL - peroxisome co - localization that was only observed in 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 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD 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 B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 localization of 3Flag - MITOL catalase in presence of NMS - 873 . Dots indicate Pearson correlation coefficient data points . In box - plots , center lines indicate medians , box limits indicate 25th 75th percentiles as determined in R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - .

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

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

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

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

O O O B-GENEPROD 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 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD 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 ) double K54 / K268 mutation did not change MITOL localization pattern . subcellular localization of MITOL K268A or K268R mutants was not drastically changed by inclusion of 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 localization of 3Flag - MITOL wild - type , K268A , or K268R mutants with catalase . Dots indicate Pearson correlation coefficient data points . In box - plots , center lines indicate medians , box limits indicate 25th 75th percentiles as determined in R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - .

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

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

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

(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 lines , 3xFlag gene cassettes were inserted upstream of MITOL stop codon using CRISPR / Cas9 - gene editing . Insertion of 3Flag - tag was verified by immunoblotting with an anti - Flag antibody . Asterisk indicates a cross - reacting band .

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

(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 ) MITOL subcellular localization was observed in MITOL - 3Flag KI HCT116 stably expressing HA - Parkin with anti - Flag , anti - catalase , 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 of boxed regions are shown in bottom panel . Scale bars , 10 µm .

O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD 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 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 localization of endogenous MITOL - 3Flag with catalase . Dots indicate Pearson correlation coefficient data points . In box - plots , center lines show medians , box limits indicate 25th 75th percentiles as determined by R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - .

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

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

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

(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 localization of endogenous MITOL PMP70 in presence of NMS - 873 . Dots indicate Pearson correlation coefficient data points . In box - plots , center lines show medians , box limits indicate 25th 75th percentiles as determined by R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - .

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

(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 stably expressing HA - Parkin were treated with valinomycin for 3 hours , collected lysates were immunoprecipitated with anti - Flag magnetic beads . immunoprecipitates were blotted using anti - Flag anti - ubiquitin antibodies . Red bars indicate ubiquitylation ; 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 mitochondria ‐ rich or peroxisome - rich fraction following fractionation . HCT116 stably expressing HA - Parkin 3Flag - MITOL were subjected to fractionation detected using anti ‐ Tom20 , anti ‐ PMP70 , anti ‐ Flag antibodies . Tom20 PMP70 were used as mitochondrial peroxisomal markers , respectively . 3 , 000 g 100 , 000 g pellets represent mitochondria ‐ rich peroxisomes - rich fractions .

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(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 ) recovery of 3Flag - MITOL between mitochondria - enriched ( 3 , 000 g pellet ) peroxisome - enriched fraction ( 100 , 000 g pellet ) following valinomycin treatment for indicated times . 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 data points . Black dots indicate of 3Flag - MITOL collected in mitochondria - enriched fractions , red dots are of 3Flag - MITOL collected in peroxisome - enriched fractions . Mean values are also shown .

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

(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 mitochondria ‐ rich or peroxisome - rich fraction following fractionation . distribution of endogenous MITOL was examined using MITOL - 3Flag knock - in HCT116 . Treatment of with valinomycin for 3 hours reduced amount of endogenous MITOL in mitochondria - enriched fraction , but concomitantly increased endogenous MITOL in 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 mitochondria ‐ rich or peroxisome - rich fraction following fractionation . distribution of endogenous MITOL was examined using MITOL - 3Flag knock - in HCT116 . Graphic data represent results of two biological replicates . In scatter plot , dots indicate data points . Black dots indicate of MITOL - 3Flag collected in mitochondria - enriched fractions , red dots are of MITOL - 3Flag collected in peroxisome - enriched fractions . Mean values are also shown .

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

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 expressing HA - Parkin wild type 3Flag - MITOL or 3Flag - MITOL lacking C - terminal 8 ( ∆ C8 ) were treated with 15 µM CCCP for 3 hours , then subjected to immunocytochemistry with anti - Flag anti - PMP70 antibodies . Expanded peroxisomes were observed in MITOL ∆ C8 - expressing . Higher magnification of boxed regions are shown in 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 E3 - inactive Cys65Ser / Cys68Ser ( CS ) H43W mutations were transfected into HeLa stably expressing HA - Parkin , treated with 15 µM CCCP , then subjected to immunocytochemistry with anti - Flag anti - PMP70 antibodies . After 3 hours of CCCP treatment , both CS H43W mutants localized on peroxisomes but expansion was not observed . Scale bars , 10 µm .

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

(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 expressing HA - Parkin MITOL wild - type or ∆ C8 were treated with 15 µM CCCP for 3 hours , number of peroxisomes was counted as PMP70 - positive dots per 100 μm2 . In box - plots , dots indicate data points , center lines show medians , box limits indicate 25th 75th percentiles as determined by R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - . abundance of peroxisomes in expressing MITOL ∆ C8 was significantly decreased as compared with expressing wild - type MITOL .

O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE 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 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 B-SUBCELLULAR O O B-GENEPROD 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 expressing HA - Parkin wild - type or ∆ C8 MITOL were treated with 15 µM CCCP for 3 hours , approximate of peroxisomes was determined as number of pixels occupied by one peroxisome . PMP70 - positive pixels per 100 µm2 were divided by number of peroxisomes in same . In box - plots , dots indicate data points , center lines show medians , box limits indicate 25th 75th percentiles as determined by R software package , whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Means number of samples are shown on box X - axis , respectively . Statistical significance was calculated using a one - Welch ' s t - .

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

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

O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-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 B-EXP_ASSAY O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-GENEPROD O 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 TF mutants gata12 wri3 grown in . Scale bar , 1 mm .

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(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 , root composite trait of root to total in - grown seedlings whether effects of TFs are dependent on condition . TFs are hierarchically clustered using Euclidean .

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(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 average relative effects of TF mutant alleles on hypocotyl , root of root to total reveal that TF lesions significantly perturbed TCA cycle - dependent growth . Mutant alleles are listed in rows traits in columns . of TF mutant alleles in heat map are colored if 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 .

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(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 , growth rate , shoot biomass , flowering time seed yield whether effects of TFs are dependent on salt treatment . TFs are hierarchically clustered using Euclidean .

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(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 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 their interactions . AGI AGI : Salt Condition terms are statistically signification ( P < 0 . 001 , P < 0 . 01 , respectively ) . Heat map under 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 , bpc4 ; circle , control condition ; triangle , 50 mM NaCl .

O O O B-TISSUE 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 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 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 along with immunocytochemistry ( ICC ) to detect SRSF1 - FLAG in U2OS . Expanded view of merged channels shows CGG foci ( arrowhead ) an example of SRSF1 - CGG foci co - localization ( arrow ) . DAPI marks 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 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 O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE 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 of CGG ( red traces ) SRSF1 ( green traces ) showing overlaps of CGG SRSF1 . ( a . u . = arbitrary unit )

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(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 with PP7 - tagged control ( partial nLUC ) to correct for non - specific binding , AUG CGG reporter RNAs . SRSF1 2 specifically immunoprecipitated with PP7 - tagged CGG reporter .

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(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 of fly eyes 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 - student ' s t 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 .

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(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 - student ' s t 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 .

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

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(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 ) 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 continued through experiment ( Log - rank Mantel - Cox ; n = 98 - 101 / genotype for Tub - GS 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 ; 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 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 B-GENEPROD 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 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 ) of fly eyes ( B ) quantitation with siRNA mediated knockdown of SRPK1 ( dSRPK1 ) ; n = 30 - 34 / genotype . Error bars represent mean + / - SD . Data information : Statistical was performed using two - Student ' s t with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

(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 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 . t - with Welch corrections for comparisons with control ; n = 31 - 34 flies / genotype . Error bars represent mean + / - SD . Data information : Statistical was performed using two - Student ' s t with Welch ' s correction , ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

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 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

(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.