Figure 1B Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint ( in hours ) after the L4 stage . Animals have been exposed to the indicated knockdown RNAi bacterial strains or a strain that only contains the empty RNAi vector . * P < 0 . 001 , Student t - test corrected for multiple comparisons at each time point using the Holm - Sidek method . N = 20 animals per genotype and timepoint . Error bars indicate SEM . C , D Co - expression via the col - 19 promoter of UbG76V - GFP ( green ) and mRFP ( red ) in C . elegans hypodermis from a single integrated transgene at L4 + 48 hours . Wild - type animals are shown . Bar , 50 microns . UbG76V - GFP fluorescence is barely detectable . E , F Co - expression via the sur - 5 promoter of UbG76V - GFP ( green ) and mCherry ( red ) in C . elegans from two separate integrated transgenes ( with focus on the intestine ) at L4 + 48 hours . Wild - type animals are shown . Bar , 50 microns . UbG76V - GFP fluorescence is barely detectable . G , H Co - expression of UbG76V - GFP ( green ) and mRFP ( red ) in C . elegans hypodermis at L4 + 48 hours in dop - 1 ( vs100 ) mutants . Bar , 50 microns . Abundant , stable UbG76V - GFP fluorescence is observed . I , J Co - expression of UbG76V - GFP ( green ) and mCherry ( red ) in C . elegans from the sur - 5 promoter at L4 + 48 hours in dop - 1 ( vs100 ) mutants . Bar , 50 microns . Abundant , stable UbG76V - GFP fluorescence is observed , particularly in the intestine . K Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint ( in hours ) after the L4 stage and of the indicated genotype . * P < 0 . 001 , Student t - test corrected for multiple comparisons at each time point using the Holm - Sidek method . N = 20 animals per genotype and timepoint . Error bars indicate SEM . L Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis of animals at L4 + 48 hours and of then indicated genotypes . * * * P < 0 . 001 , ANOVA with Dunnetts posthoc comparison to the wild - type control . N = 20 animals per genotype and timepoint . Error bars indicate SEM . M Quantified fluorescence of UbG76V - GFP normalized to mCherry ( both expressed by the sur - 5 promoter ) in the intestine of animals at L4 + 48 hours and of then indicated genotypes . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to the wild - type control . N = 20 animals per genotype and timepoint . Error bars indicate SEM .

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

Figure 1B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage. Animals have been exposed to the indicated knockdown RNAi bacterial strains or a strain that only contains the empty RNAi vector. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM.C,D Co-expression via the col-19 promoter of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis from a single integrated transgene at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.E,F Co-expression via the sur-5 promoter of UbG76V-GFP (green) and mCherry (red) in C. elegans from two separate integrated transgenes (with focus on the intestine) at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.G,H Co-expression of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed.I,J Co-expression of UbG76V-GFP (green) and mCherry (red) in C. elegans from the sur-5 promoter at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed, particularly in the intestine.K Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage and of the indicated genotype. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM.L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals at L4+48 hours and of then indicated genotypes. ***P<0.001, ANOVA with Dunnetts posthoc comparison to the wild-type control. N=20 animals per genotype and timepoint. Error bars indicate SEM.M Quantified fluorescence of UbG76V-GFP normalized to mCherry (both expressed by the sur-5 promoter) in the intestine of animals at L4+48 hours and of then indicated genotypes. ***P<0.001, **P<0.01, *P<0.05, ANOVA with Dunnetts posthoc comparison to the wild-type control. N=20 animals per genotype and timepoint. Error bars indicate SEM.


Figure 2A Western blot of lysed nematodes at the L4 + 48 hour stage , probed with antibodies recognizing , ubiquitin , GFP , RPN - 11 , multiple 20S proteasome subunits , or actin as a loading control . Animals express UbG76V - GFP protein in their hypodermis . The position of molecular weight markers is shown on the left of each blot . The position of UbG76V - GFP protein , as well as UbG76V - GFP with the indicated number of additional ubiquitin moieties , based on molecular weight , is indicated on the right of each blot . Twenty animals were loaded per lane for each indicated genotype ( WT indicates wild type ) . B Western blot of lysed nematodes at the L4 + 48 hour stage , probed and annotated as in ( A ) . Animals express UbG76V - GFP protein from the sur - 5 promoter . Thirty animals were loaded per lane for each indicated genotype ( WT indicates wild type ) . C Quantified fluorescence of UbG76V - GFP normalized to mRFP from lysates of transgenic nematodes at L4 + 48 hours that express these reporters in the hypodermis . Animals were exposed to bacteria containing the indicated RNAi clone or an empty RNAi vector as a control . * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control . N = 3 trials . Error bars indicate SEM . D Quantified epoxomicin - sensitive proteasome activity ( as measured through the turnover of fluorescent chymotrypsin substrate ) from the same lysates and exposed to the same RNAi as in ( C ) . * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control . N = 3 trials . Error bars indicate SEM . E Quantified fluorescence of UbG76V - GFP normalized to mRFP from lysates of transgenic nematodes at L4 + 48 hours that express these reporters in the hypodermis . Animals were of the indicated genotype . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to wild type . N = 4 trials . Error bars indicate SEM . F Quantified epoxomicin - sensitive proteasome activity ( as measured through the turnover of fluorescent chymotrypsin substrate ) from the same lysates as in ( E ) . No statistical difference ( P < 0 . 05 cutoff ) was detected by ANOVA . N = 4 trials . G Percentage of live animals of the indicated genotype assayed after 6 - hour exposure to heat shock . 100 % of control animals of the same genotype but not given heat shock survived . * P < 0 . 01 , Student t - test . N = 3 trials . Error bars indicate SEM . H Percent of animals of the indicated genotype containing aggregates of Poly ( Q ) 44 : : YFP at the indicated time ( in days ) after L4 stage . * P < 0 . 01 , Student t - test . N = 3 trials , 50 - 80 animals per trial per day . Error bars indicate SEM .

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

Figure 2A Western blot of lysed nematodes at the L4+48 hour stage, probed with antibodies recognizing, ubiquitin, GFP, RPN-11, multiple 20S proteasome subunits, or actin as a loading control. Animals express UbG76V-GFP protein in their hypodermis. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated on the right of each blot. Twenty animals were loaded per lane for each indicated genotype (WT indicates wild type).B Western blot of lysed nematodes at the L4+48 hour stage, probed and annotated as in (A). Animals express UbG76V-GFP protein from the sur-5 promoter. Thirty animals were loaded per lane for each indicated genotype (WT indicates wild type).C Quantified fluorescence of UbG76V-GFP normalized to mRFP from lysates of transgenic nematodes at L4+48 hours that express these reporters in the hypodermis. Animals were exposed to bacteria containing the indicated RNAi clone or an empty RNAi vector as a control. *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM.D Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates and exposed to the same RNAi as in (C). *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM.E Quantified fluorescence of UbG76V-GFP normalized to mRFP from lysates of transgenic nematodes at L4+48 hours that express these reporters in the hypodermis. Animals were of the indicated genotype. ***P<0.001, **P<0.01, *P<0.05, ANOVA with Dunnetts posthoc comparison to wild type. N=4 trials. Error bars indicate SEM.F Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates as in (E). No statistical difference (P<0.05 cutoff) was detected by ANOVA. N=4 trials.G Percentage of live animals of the indicated genotype assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. *P<0.01, Student t-test. N=3 trials. Error bars indicate SEM.H Percent of animals of the indicated genotype containing aggregates of Poly(Q)44::YFP at the indicated time (in days) after L4 stage. *P<0.01, Student t-test. N=3 trials, 50-80 animals per trial per day. Error bars indicate SEM.


Figure 3A Relative abundance of mRNA for the indicated xenobiotic stress response gene ( or UbG76V - GFP or mRFP ) detected in wild type versus dop - 1 mutants . Values are based on RNA - seq / EdgeR analysis , with the relative levels for each gene normalized to the value observed in the wild - type control . * * * * P < 0 . 0001 , * * P < 0 . 01 , * P < 0 . 05 FDR from EdgeR analysis using a Benjamini and Hochberg correction . B , C Quantified fluorescence of UbG76V - GFP normalized to ( B ) mRFP in the hypodermis from L4 + 48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA , Tukey ' s multiple comparison test compared to control . N = 20 animals per genotype and timepoint . Error bars indicate SEM . B , C Quantified fluorescence of UbG76V - GFP normalized to ( C ) mCherry in the intestine from L4 + 48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA , Tukey ' s multiple comparison test compared to control . N = 20 animals per genotype and timepoint . Error bars indicate SEM . D Percentage of live animals raised on the indicated feeding RNAi ( or empty vector control ) bacteria assayed after 6 - hour exposure to heat shock . 100 % of control animals of the same genotype but not given heat shock survived . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , ANOVA , Tukey ' s multiple comparison test compared to wild type . N = 3 trials . Error bars indicate SEM . E Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from L4 + 48 hour animals of the indicated genotype . * * * * P < 0 . 0001 , ANOVA , Tukey ' s multiple comparison test compared to wild type . N = 20 animals per genotype and timepoint . Error bars indicate SEM . F Percentage of live animals of the indicated genotype assayed after 6 - hour exposure to heat shock . 100 % of control animals of the same genotype but not given heat shock survived . * * P < 0 . 01 , * P < 0 . 05 , ANOVA , Tukey ' s multiple comparison test compared to wild type . N = 3 trials . Error bars indicate SEM . G Percent of animals of the indicated genotype containing aggregates of Poly ( Q ) 44 : : YFP at the indicated time ( in days ) after L4 stage . * P < 0 . 001 , Student t - test corrected for multiple comparisons at each time point using the Holm - Sidek method . N = 3 trials , 50 - 80 animals per trial per day . Error bars indicate SEM .

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

Figure 3A Relative abundance of mRNA for the indicated xenobiotic stress response gene (or UbG76V-GFP or mRFP) detected in wild type versus dop-1 mutants. Values are based on RNA-seq/EdgeR analysis, with the relative levels for each gene normalized to the value observed in the wild-type control. ****P<0.0001, **P<0.01, *P<0.05 FDR from EdgeR analysis using a Benjamini and Hochberg correction.B,C Quantified fluorescence of UbG76V-GFP normalized to (B) mRFP in the hypodermis from L4+48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to control. N=20 animals per genotype and timepoint. Error bars indicate SEM.B,C Quantified fluorescence of UbG76V-GFP normalized to (C) mCherry in the intestine from L4+48 hour animals exposed to feeding RNAi for either an empty vector control or the indicated xenobiotic stress gene. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to control. N=20 animals per genotype and timepoint. Error bars indicate SEM.D Percentage of live animals raised on the indicated feeding RNAi (or empty vector control) bacteria assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. ***P<0.001, **P<0.01, *P<0.05, ANOVA, Tukey's multiple comparison test compared to wild type. N=3 trials. Error bars indicate SEM.E Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from L4+48 hour animals of the indicated genotype. ****P<0.0001, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype and timepoint. Error bars indicate SEM.F Percentage of live animals of the indicated genotype assayed after 6-hour exposure to heat shock. 100% of control animals of the same genotype but not given heat shock survived. **P<0.01, *P<0.05, ANOVA, Tukey's multiple comparison test compared to wild type. N=3 trials. Error bars indicate SEM.G Percent of animals of the indicated genotype containing aggregates of Poly(Q)44::YFP at the indicated time (in days) after L4 stage. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=3 trials, 50-80 animals per trial per day. Error bars indicate SEM.


Figure 4A Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from L4 + 48 hour animals of the indicated genotype . * * * * P < 0 . 0001 , ANOVA , Tukey ' s multiple comparison test compared to wild type . N = 20 animals per genotype . Error bars indicate SEM . B Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from either L4 + 24 hour animals ( left half of graph ) or L4 + 48 hour animals ( right half of graph ) of the indicated genotype . Animals were either continually grown on regular OP50 food ( gray bars ) or switched to PA14 food at the L4 stage ( orange bars ) . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA , Tukey ' s multiple comparison test compared to wild type . N = 20 animals per genotype . Error bars indicate SEM . C Survival curves ( percentage of live animals assayed each hour after PA14 exposure ) for the indicated mutants at 20oC . Plog - rank < 0 . 0001 . Median survival time was 90 hours for wildtype ( N = 304 ) , 72 hours for dop - 1 ( vs100 ) ( N = 180 ) , 78 hours for elt - 3 ( gk121 ) ( N = 120 ) , 78 hours for pqm - 1 ( ok485 ) ( N = 116 ) , 78 hours for nhr - 28 ( gk568 ) ( N = 123 ) , and 42 hours for skn - 1 ( tm3411 ) ( N = 33 ) . Eight wild - type animals and six dop - 1 mutants were censored ( the animals crawled up the side of the plate and dessicated ) . The media contained 50 µg / ml of 5 - fluoro - 2 ′ - deoxyuridine ( FUdR to prevent the growth of egg offspring during the experiment . D Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from either L4 + 48 hour animals ( left half of graph ) or L4 + 72 hour animals ( right half of graph ) of the indicated genotype . Animals were either continually grown on regular OP50 food ( gray bars ) or switched to NGM plates containing 1 mM paraquat at the L4 stage ( orange bars ) . * * * * P < 0 . 0001 , * * * P < 0 . 001 , ANOVA , Tukey ' s multiple comparison test compared to wild type . # P < 0 . 001 ANOVA , Bonferoni multicomparison as indicated by the bracketed lines . N = 20 animals per genotype . Error bars indicate SEM . No UbG76V - GFP fluorescence was detected in wild type at L4 + 72 hours on either OP50 or switched to NGM plates containing 1mM paraquat .

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

Figure 4A Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from L4+48 hour animals of the indicated genotype. ****P<0.0001, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype. Error bars indicate SEM.B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+24 hour animals (left half of graph) or L4+48 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to PA14 food at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, **P<0.01, ANOVA, Tukey's multiple comparison test compared to wild type. N=20 animals per genotype. Error bars indicate SEM.C Survival curves (percentage of live animals assayed each hour after PA14 exposure) for the indicated mutants at 20oC. Plog-rank<0.0001. Median survival time was 90 hours for wildtype (N=304), 72 hours for dop-1(vs100) (N=180), 78 hours for elt-3(gk121) (N=120), 78 hours for pqm-1(ok485) (N=116), 78 hours for nhr-28(gk568) (N=123), and 42 hours for skn-1(tm3411) (N=33). Eight wild-type animals and six dop-1 mutants were censored (the animals crawled up the side of the plate and dessicated). The media contained 50 µg/ml of 5-fluoro-2′-deoxyuridine (FUdR to prevent the growth of egg offspring during the experiment.D Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+48 hour animals (left half of graph) or L4+72 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to NGM plates containing 1 mM paraquat at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, ANOVA, Tukey's multiple comparison test compared to wild type. #P<0.001 ANOVA, Bonferoni multicomparison as indicated by the bracketed lines. N=20 animals per genotype. Error bars indicate SEM. No UbG76V-GFP fluorescence was detected in wild type at L4+72 hours on either OP50 or switched to NGM plates containing 1mM paraquat.


Figure 1A : Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a screen of 23 pediatric cancer cell lines . Rhabdoid tumor cell lines ( red ) cluster towards the left of the graph indicating these cell lines are more sensitive to mithramycin . These results confirm a previously published screen ( Osgood et al . , 2016 ) . B : Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a published screen of 445 agents in 62 sarcoma cell lines ( Teicher et al . , 2015 ) . The rhabdoid tumor cell line , G401 ( red ) , appears on the left side of the graph indicating this cell line is more sensitive to mithramycin . C : Dose response curves of rhabdoid tumor and Ewing sarcoma cell lines . RT cell lines ( black ) are sensitive to mithramycin treatment with a similar IC50 value as TC32 ES cells ( grey ) . RT cell lines are not sensitive to three broadly - active chemotherapeutic agents : etoposide , doxorubicin or SN38 . Data represents mean with standard deviation derived from three independent experiments .

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

Figure 1A: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a screen of 23 pediatric cancer cell lines. Rhabdoid tumor cell lines (red) cluster towards the left of the graph indicating these cell lines are more sensitive to mithramycin. These results confirm a previously published screen (Osgood et al., 2016).B: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a published screen of 445 agents in 62 sarcoma cell lines (Teicher et al., 2015). The rhabdoid tumor cell line, G401 (red), appears on the left side of the graph indicating this cell line is more sensitive to mithramycin.C: Dose response curves of rhabdoid tumor and Ewing sarcoma cell lines. RT cell lines (black) are sensitive to mithramycin treatment with a similar IC50 value as TC32 ES cells (grey). RT cell lines are not sensitive to three broadly-active chemotherapeutic agents: etoposide, doxorubicin or SN38. Data represents mean with standard deviation derived from three independent experiments.


Figure 2A , B : Western blot showing concentration dependent increase in γH2AX following 8 - hour exposure to etoposide in BT12 and G401 RT cells ( A ) . Red bar indicates the measured IC50 ( Figure 1C ) . Despite induction of DNA damage , 15 μM etoposide does not lead to apoptosis as indicated by live cell imaging in the presence of cleaved caspase 3 , 7 ( CC3 , 7 ) reagent that fluoresces with caspase activation ( B ) . Scale bar ( lower left ) : 150μm . C , D : Western blot showing concentration dependent increase in γH2AX following 8 - hour exposure to mithramycin ( MMA ) in BT12 and G401 RT cells ( C ) . Red bar indicates the measured IC50 ( Figure 1C ) . 100 nM Mithramycin induces apoptosis at 8 - hours as measured by CC3 , 7 fluorescence without the presence of DNA damage ( D ) . Scale bar ( lower left ) : 150μm . E : BT12 cells treated with 20nM MMA show a different cellular phenotype than with 100nM . Beyond 24h of exposure , there is evidence of mesenchymal differentiation and the appearance of maturing adipocytes . Scale bar ( lower left ) : 150μm . F , G : Western blot showing concentration dependent increase in γH2AX following 8 - hour exposure to EC8042 in BT12 and G401 RT cells . Red bar indicates the measured IC50 ( Figure 1C ) . In contrast to apoptotic induction , 75nM EC8042 exhibits evidence of mesenchymal differentiation , similar to 20nM MMA ( E , G ) . Please note , solvent control for ( E ) and ( G ) is the same although different fields are shown . Scale bar ( lower left ) : 150μm . H : Mesenchymal differentiation confirmed with oil red O staining of lipid deposits following 20nM MMA or 75nM EC8042 treatment for 24 and 48 - hours . Please note , solvent control for 20nM MMA and 75nM EC8042 is the same . Scale bar ( lower left ) : 150μm . I : PPARγ mRNA expression is induced following 20nM mithramycin treatment as measured by qPCR fold - change relative to GAPDH ( 2ddCT ) . * * , P = 0 . 002 , * * * * , P = 0 . 0001 . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons .

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

Figure 2A, B: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to etoposide in BT12 and G401 RT cells (A). Red bar indicates the measured IC50 (Figure 1C). Despite induction of DNA damage, 15 μM etoposide does not lead to apoptosis as indicated by live cell imaging in the presence of cleaved caspase 3,7 (CC3,7) reagent that fluoresces with caspase activation (B). Scale bar (lower left): 150μm.  C, D: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to mithramycin (MMA) in BT12 and G401 RT cells (C). Red bar indicates the measured IC50 (Figure 1C). 100 nM Mithramycin induces apoptosis at 8-hours as measured by CC3,7 fluorescence without the presence of DNA damage (D). Scale bar (lower left): 150μm.  E: BT12 cells treated with 20nM MMA show a different cellular phenotype than with 100nM. Beyond 24h of exposure, there is evidence of mesenchymal differentiation and the appearance of maturing adipocytes. Scale bar (lower left): 150μm.  F, G: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to EC8042 in BT12 and G401 RT cells. Red bar indicates the measured IC50 (Figure 1C). In contrast to apoptotic induction, 75nM EC8042 exhibits evidence of mesenchymal differentiation, similar to 20nM MMA (E,G). Please note, solvent control for (E) and (G) is the same although different fields are shown. Scale bar (lower left): 150μm.  H: Mesenchymal differentiation confirmed with oil red O staining of lipid deposits following 20nM MMA or 75nM EC8042 treatment for 24 and 48-hours. Please note, solvent control for 20nM MMA and 75nM EC8042 is the same. Scale bar (lower left): 150μm.  I: PPARγ mRNA expression is induced following 20nM mithramycin treatment as measured by qPCR fold-change relative to GAPDH (2ddCT). **, P = 0.002, ****, P = 0.0001. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.


Figure 3 A , B : Mithramycin displaces SMARCC1 and SMARCE1 SWI / SNF subunits from chromatin in a time - dependent manner in BT12 rhabdoid tumor ( A ) but not U20S osteosarcoma ( B ) cells . Western blot analysis showing whole cell lysate ( Total ) , cytoplasmic soluble ( CS ) , nuclear soluble ( NS ) , and chromatin bound ( Chr ) fractions collected after exposure to solvent ( S ) or 100nM mithramycin for 8 or 18h and probed for the SWI / SNF subunits ( BRD9 , SMARCC1 or SMARCE1 ) or H3 ( chromatin fraction control ) and GAPDH ( soluble fraction control ) . C : SMARCC1 and SMARCE1 mRNA expression does not change following 100nM mithramycin treatment as measured by qPCR fold - change relative to GAPDH ( 2ddCT ) . Data represents mean with standard deviation derived from three independent experiments . D : Addition of the proteasome inhibitor ( bortezomib ) rescues the loss of protein expression following 8 or 18 - hour mithramycin treatment . BT12 cells were treated for 8 or 18 - hours with solvent ( S ) , mithramycin ( M , 100nM ) , bortezomib ( PI , 2 . 5μM ) , or combination of 100nM mithramycin and 2 . 5μM bortezomib ( C ) . E : Loss of SWI / SNF occupancy at defined loci in the genome as measure by chromatin immunoprecipitation qPCR ( ChIP - qPCR ) at previously described SWI / SNF target genes , MYT1 ( 8 - hour , P = 0 . 0001 ; 18 - hour P = 0 . 0001 ) and CCND1 ( 8 - hour , P = 0 . 0001 ; 18 - hour P = 0 . 0001 ) . ChIP quantitation is percent input ( ng of immunoprecipitated DNA / input DNA * 100 ) determined by absolute quantitation of sheared chromatin relative to a standard curve . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons . F : Chromatin immunoprecipitation qPCR ( ChIP - qPCR ) of H3K27me3 at MYT1 ( 8 - hour , P = 0 . 0001 ; 18 - hour P = 0 . 0001 ) and CCND1 ( 8 - hour , P = 0 . 02 ; 18 - hour P = 0 . 0001 ) . H3K27me3 occupancy is increased in a time - dependent manner . ChIP quantitation is percent input ( ng of immunoprecipitated DNA / input DNA * 100 ) determined by absolute quantitation of sheared chromatin relative to a standard curve . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons . Western blot showing concentration - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in BT12 cells relative to loading control ( H3 ) . Western blot showing concentration - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in G401 cells relative to loading control ( H3 ) . Re - expression of SMARCB1 following doxycycline treatment inhibits the mithramycin - dependent effects on H3K27me3 amplification

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

Figure 3 A, B: Mithramycin displaces SMARCC1 and SMARCE1 SWI/SNF subunits from chromatin in a time-dependent manner in BT12 rhabdoid tumor (A) but not U20S osteosarcoma (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin bound (Chr) fractions collected after exposure to solvent (S) or 100nM mithramycin for 8 or 18h and probed for the SWI/SNF subunits (BRD9, SMARCC1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control). C: SMARCC1 and SMARCE1 mRNA expression does not change following 100nM mithramycin treatment as measured by qPCR fold-change relative to GAPDH (2ddCT). Data represents mean with standard deviation derived from three independent experiments. D: Addition of the proteasome inhibitor (bortezomib) rescues the loss of protein expression following 8 or 18-hour mithramycin treatment. BT12 cells were treated for 8 or 18-hours with solvent (S), mithramycin (M, 100nM), bortezomib (PI, 2.5μM), or combination of 100nM mithramycin and 2.5μM bortezomib (C).  E: Loss of SWI/SNF occupancy at defined loci in the genome as measure by chromatin immunoprecipitation qPCR (ChIP-qPCR) at previously described SWI/SNF target genes, MYT1 (8-hour, P=0.0001; 18-hour P=0.0001) and CCND1 (8-hour, P=0.0001; 18-hour P=0.0001). ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.  F: Chromatin immunoprecipitation qPCR (ChIP-qPCR) of H3K27me3 at MYT1 (8-hour, P= 0.0001; 18-hour P=0.0001) and CCND1 (8-hour, P=0.02; 18-hour P=0.0001). H3K27me3 occupancy is increased in a time-dependent manner. ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in BT12 cells relative to loading control (H3). Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in G401 cells relative to loading control (H3). Re-expression of SMARCB1 following doxycycline treatment inhibits the mithramycin-dependent effects on H3K27me3 amplification


Figure 4 A , B : Mithramycin displaces BRD9 and SMARCE1 SWI / SNF subunits from chromatin in a time - dependent manner in G401 ( A ) but only BRD9 with SMARCB1 re - expression in G401 ( B ) cells . Western blot analysis showing whole cell lysate ( Total ) , cytoplasmic soluble ( CS ) , nuclear soluble ( NS ) , and chromatin bound ( Chr ) fractions collected after exposure to solvent ( S ) or 100nM mithramycin for 8 or 18h and probed for the SWI / SNF subunits ( BRD9 , SMARCB1 or SMARCE1 ) or H3 ( chromatin fraction control ) and GAPDH ( soluble fraction control ) . C : BT12 cells show a threshold of exposure that leads to irreversible growth inhibition . The cells were exposed to 100nM MMA for the indicated times followed by replacement with drug - free medium . Beyond 8h ( red ) of mithramycin exposure , the cells do not recover proliferative potential and exhibit a phenotype consistent with cell death . Data represents mean with standard deviation derived from three independent experiments . D : Mithramycin leads to H3K27me3 amplification in a time - dependent manner that precedes the induction of apoptosis as measured by the cleavage of PARP . Western blot lysates collected at 2h , 4h , 6h , 8h , 12h , 18h , 24h of continuous 100nM mithramycin treatment . E : Suppression of EZH2 expression and activity antagonizes mithramycin activity in BT12 rhabdoid tumor cells . Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the PRC2 subunit EZH2 or treatment with the EZH2 inhibitor EPZ - 6438 relative to mithramycin alone ( media ) or a non - targeting siRNA ( siNeg ) . Data represents mean with standard deviation derived from three independent experiments . F : Suppression of SMARCB1 sensitizes U2OS osteosarcoma cells ( wild type SWI / SNF ) to mithramycin . Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the SWI / SNF subunit SMARCB1 relative to a non - targeting siRNA ( siNeg ) . Data represents mean with standard deviation derived from three independent experiments . G . Western blot showing concentration - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in SMARCB1 - silenced U2OS cells relative to loading control ( H3 ) . Knockdown of SMARCB1 following siRNA suppression triggers mithramycin - dependent H3K27me3 amplification , while siNegative ( control ) does not have an effect on H3K27me3 following mithramycin exposure .

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

Figure 4 A, B: Mithramycin displaces BRD9 and SMARCE1 SWI/SNF subunits from chromatin in a time-dependent manner in G401 (A) but only BRD9 with SMARCB1 re-expression in G401 (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin bound (Chr) fractions collected after exposure to solvent (S) or 100nM mithramycin for 8 or 18h and probed for the SWI/SNF subunits (BRD9, SMARCB1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control).  C: BT12 cells show a threshold of exposure that leads to irreversible growth inhibition. The cells were exposed to 100nM MMA for the indicated times followed by replacement with drug-free medium. Beyond 8h (red) of mithramycin exposure, the cells do not recover proliferative potential and exhibit a phenotype consistent with cell death. Data represents mean with standard deviation derived from three independent experiments.  D: Mithramycin leads to H3K27me3 amplification in a time-dependent manner that precedes the induction of apoptosis as measured by the cleavage of PARP. Western blot lysates collected at 2h, 4h, 6h, 8h, 12h, 18h, 24h of continuous 100nM mithramycin treatment.  E: Suppression of EZH2 expression and activity antagonizes mithramycin activity in BT12 rhabdoid tumor cells. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the PRC2 subunit EZH2 or treatment with the EZH2 inhibitor EPZ-6438 relative to mithramycin alone (media) or a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments.  F: Suppression of SMARCB1 sensitizes U2OS osteosarcoma cells (wild type SWI/SNF) to mithramycin. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the SWI/SNF subunit SMARCB1 relative to a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments.  G. Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in SMARCB1- silenced U2OS cells relative to loading control (H3). Knockdown of SMARCB1 following siRNA suppression triggers mithramycin-dependent H3K27me3 amplification, while siNegative (control) does not have an effect on H3K27me3 following mithramycin exposure.


Figure 5A - C : Volcano plot showing gene expression trends at 8h MMA treatment . Dashed lines represent a 2 log2FC ( drug over control ) ( A ) or 1 . 5 log2FC ( drug over control ) ( B ) and 10e - 7 q - value threshold . Quantification of induced and repressed genes in ( C ) . Labels indicate names of genes that meet the logFC and q - value threshold . D - F : Volcano plot showing gene expression trends at 18h MMA treatment . Dashed lines represent a 2 log2FC ( drug over control ) ( D ) or 1 . 5 log2FC ( drug over control ) ( E ) and 10e - 7 q - value threshold . Quantification of induced and repressed genes in ( F ) . Labels indicate names of genes that meet the logFC and q - value threshold . G : SP1 protein expression is reduced following mithramycin exposure . Western blot showing suppression of SP1 expression compared to loading control ( GAPDH ) after 100nM mithramycin exposure for 1h , 4h , 8h , 12h , 18h . The loss of SP1 expression is associated with a decrease in SWI / SNF occupancy of the SP1 promoter and an increase in H3K27me3 . Data represents ChIP - qPCR analysis following 8 - hours or 18 - hours of 100nM mithramycin exposure and immunoprecipitation of SMARCC1 ( 8 - hour , P = 0 . 0006 ; 18 - hour P = 0 . 0001 ) . ChIP quantitation is percent input ( ng of immunoprecipitated DNA / input DNA * 100 ) determined by absolute quantitation of sheared chromatin relative to a standard curve . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons . I : The loss of SP1 expression is associated with a decrease in SWI / SNF occupancy of the SP1 promoter and an increase in H3K27me3 . Data represents ChIP - qPCR analysis following 8 - hours or 18 - hours of 100nM mithramycin exposure and immunoprecipitation of H3K27me3 ( 8 - hour , P = 0 . 009 ; 18 - hour P = 0 . 0001 ) ChIP quantitation is percent input ( ng of immunoprecipitated DNA / input DNA * 100 ) determined by absolute quantitation of sheared chromatin relative to a standard curve . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons .

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

Figure 5A-C: Volcano plot showing gene expression trends at 8h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (A) or 1.5 log2FC (drug over control) (B) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (C). Labels indicate names of genes that meet the logFC and q-value threshold.D-F: Volcano plot showing gene expression trends at 18h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (D) or 1.5 log2FC (drug over control) (E) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (F). Labels indicate names of genes that meet the logFC and q-value threshold. G: SP1 protein expression is reduced following mithramycin exposure. Western blot showing suppression of SP1 expression compared to loading control (GAPDH) after 100nM mithramycin exposure for 1h, 4h, 8h, 12h, 18h.  The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of SMARCC1 (8-hour, P= 0.0006; 18-hour P=0.0001) . ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.  I: The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of H3K27me3 (8-hour, P= 0.009; 18-hour P=0.0001) ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.


Figure 6A : Heatmaps depicting ATAC - seq global chromatin structure following 8 - hours ( middle ) and 18 - hours ( right ) 100nM MMA treatment . A 2kb window is centered on the TSS . Chromatin accessibility clusters at the TSS and does not change globally relative to solvent . Quantification of genes that gain accessibility or reduce accessibility on the right . E : IGV tracks of rhabdoid tumor genes previously identified to be occupied by non - canonical SWI / SNF ( Michel et al . , 2018 ) . ID3 and JUND3 decrease in H3K27ac occupancy and chromatin accessibility following exposure to mithramycin for 8 - hours or 18 - hours . F : IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI / SNF loss ( Erkek et al . , 2019 ) . CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8 - hours and 18 - hours mithramycin treatment . G : IGV tracks of genes identified to have an increase in accessibility , H3K27ac and gene expression following mithramycin exposure . BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation , respectively .

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

Figure 6A: Heatmaps depicting ATAC-seq global chromatin structure following 8-hours (middle) and 18-hours (right) 100nM MMA treatment. A 2kb window is centered on the TSS. Chromatin accessibility clusters at the TSS and does not change globally relative to solvent. Quantification of genes that gain accessibility or reduce accessibility on the right. E: IGV tracks of rhabdoid tumor genes previously identified to be occupied by non-canonical SWI/SNF (Michel et al., 2018). ID3 and JUND3 decrease in H3K27ac occupancy and chromatin accessibility following exposure to mithramycin for 8-hours or 18-hours.  F: IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI/SNF loss (Erkek et al., 2019). CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8-hours and 18-hours mithramycin treatment.  G: IGV tracks of genes identified to have an increase in accessibility, H3K27ac and gene expression following mithramycin exposure. BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation, respectively.


Figure 7 A : Immunohistochemistry analysis recapitulates the biochemistry described in vitro for mithramycin . G401 tumor sections at 20X magnification stained with H & E , cleaved caspase 3 ( CC3 ; apoptosis ) or H3K27me3 . A marked increase in CC3 that correlates with H3K27me3 staining is seen only in mice treated with the continuous infusion schedule but not vehicle . Scale bar ( lower left ) : 50μm . B : Prolonged durable response and cure of mice bearing G401 xenografts treated with 30mg / kg EC8042 administered continuously over 72 - hours . Treatment duration indicated by gray shaded box . Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity ( see text ) . C : Kaplan - Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3 - day continuous infusions of EC8042 in ( B ) . The shaded box indicates the duration of treatment . Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity ( see text ) . D : 20X image of section of G401 treated tumors stained for H3K27me3 , cleaved caspase 3 , and γH2AX . The sections compare vehicle to treatment started on day 1 with 30 mg / kg of EC8042 administered continuously for 72 - hours ( 3 - day pump ) . H3K27me3 increases and correlates with apoptosis ( CC3 ) , however induction of CC3 is modest . γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects . Scale bar ( lower left ) : 50μm .

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

Figure 7 A: Immunohistochemistry analysis recapitulates the biochemistry described in vitro for mithramycin. G401 tumor sections at 20X magnification stained with H&E, cleaved caspase 3 (CC3; apoptosis) or H3K27me3. A marked increase in CC3 that correlates with H3K27me3 staining is seen only in mice treated with the continuous infusion schedule but not vehicle. Scale bar (lower left): 50μm.  B: Prolonged durable response and cure of mice bearing G401 xenografts treated with 30mg/kg EC8042 administered continuously over 72-hours. Treatment duration indicated by gray shaded box. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).  C: Kaplan-Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3-day continuous infusions of EC8042 in (B). The shaded box indicates the duration of treatment. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).  D: 20X image of section of G401 treated tumors stained for H3K27me3, cleaved caspase 3, and γH2AX. The sections compare vehicle to treatment started on day 1 with 30 mg/kg of EC8042 administered continuously for 72-hours (3-day pump). H3K27me3 increases and correlates with apoptosis (CC3), however induction of CC3 is modest. γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects. Scale bar (lower left): 50μm.


Figure 8 Immunohistochemistry analysis of H & E stains from G401 xenograft tumors on 1 , 3 , and 7 - days after treatment with vehicle or 3 - day EC8042 infusion . EC8042 treated xenograft tumors exhibit evidence of mesenchymal differentiation compared to vehicle . microCT analysis of xenograft tumors on 7 - days after treatment exhibit enhanced calcification compared to vehicle . IHC scale bar ( lower left ) : 100μm .

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

Figure 8 Immunohistochemistry analysis of H&E stains from G401 xenograft tumors on 1, 3, and 7-days after treatment with vehicle or 3-day EC8042 infusion. EC8042 treated xenograft tumors exhibit evidence of mesenchymal differentiation compared to vehicle. microCT analysis of xenograft tumors on 7-days after treatment exhibit enhanced calcification compared to vehicle. IHC scale bar (lower left): 100μm.


Figure 1B . RNAseq data are plotted in log2 fold change ( FC ) levels of protrusions over cell bodies against adjusted - log10 false discovery rate ( FDR ) ( n = 2 replicates , average FC values are represented ) . The horizontal dashed line marks the FDR ( q ) = 0 . 05 threshold ; vertical dashed lines mark the FC = 0 . 625 ( left ) or FC = 1 . 6 ( right ) thresholds . C . Heat map represents the k - means clustering of transcript log2 FC levels ( protrusions over cell bodies ) extracted from RNAseq datasets published elsewhere . The corresponding HUVEC FC levels are shown in parallel . D . smFISH co - detection of k 5 mRNAs and GAPDH in subconfluent motile HUVECs . arrows indicate orientation of RNA localisation ; yellow dashed lines outline cell borders ; red circles highlight smFISH spots ; scale bars = 20 µmE . Polarisation Index ( PI ) of k 5 mRNAs and GAPDH in co - detected in HUVECs ( n ≥ 28 cells ; * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Wilcoxon test ) . F . k 5 mRNAs PIs plotted against respective GAPDH PIs . The slope of the coloured lines represents the average k 5 mRNA / GAPDH PI ratio ; the dashed grey line represents a 1 : 1 ratio ( n ≥ 28 cells ) . G . Top : distribution pattern of mRNAs clustered in k 2 , k 5 , k 7 and GAPDH . Bottom : smFISH co - detection of exemplar k7 / k 2 mRNAs and GAPDH in subconfluent motile HUVECs . arrows indicate orientation of RNA localisation ; yellow dashed lines outline cell borders ; red circles highlight smFISH spots ; scale bars = 20 µmH . PIs of k 7 mRNAs and GAPDH co - detected in HUVECs ( n ≥ 25 cells ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; paired t test ) . I . PIs of k 2 mRNAs and GAPDH co - detected in HUVECs ( n ≥ 19 cells ; * P < 0 . 05 ; Wilcoxon test ) .

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

Figure 1B. RNAseq data are plotted in log2 fold change (FC) levels of protrusions over cell bodies against adjusted -log10 false discovery rate (FDR) (n=2 replicates, average FC values are represented). The horizontal dashed line marks the FDR (q) = 0.05 threshold; vertical dashed lines mark the FC = 0.625 (left) or FC = 1.6 (right) thresholds.C. Heat map represents the k-means clustering of transcript log2 FC levels (protrusions over cell bodies) extracted from RNAseq datasets published elsewhere. The corresponding HUVEC FC levels are shown in parallel.D. smFISH co-detection of k 5 mRNAs and GAPDH in subconfluent motile HUVECs. arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µmE. Polarisation Index (PI) of k 5 mRNAs and GAPDH in co-detected in HUVECs (n≥28 cells; **P<0.01, ***P<0.001, ****P<0.0001; Wilcoxon test).F. k 5 mRNAs PIs plotted against respective GAPDH PIs. The slope of the coloured lines represents the average k 5 mRNA/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n≥28 cells).G. Top: distribution pattern of mRNAs clustered in k 2, k 5, k 7 and GAPDH. Bottom: smFISH co-detection of exemplar k7 / k 2 mRNAs and GAPDH in subconfluent motile HUVECs. arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µmH. PIs of k 7 mRNAs and GAPDH co-detected in HUVECs (n≥25 cells; *P<0.05, **P<0.01, ***P<0.001; paired t test).I. PIs of k 2 mRNAs and GAPDH co-detected in HUVECs (n≥19 cells; *P<0.05; Wilcoxon test).


Figure 2E . Left : representative subconfluent motile cells co - transfected with plasmids expressing Lyn - mCherry , MCP - GFPnls and 24xMS2 - RAB13 3 ' UTR or 24xMS2 . Right : percentage of cells with MCP - GFP localised to protrusions when co - transfected with full length or deletion versions of RAB13 3 ' UTR ( n ≥ 3 experiments ) . white arrowheads indicate non - nuclear localisation of MCP - GFPnls ; arrows indicate the orientation of RNA localisation ; yellow dashed lines outline cell borders ; scale bars = 20 µm For each k 5 mRNA 3 ' UTR , a diagram of the full length 3 ' UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions ; black arrowheads indicate predicted polyadenylation sitesF . Left : representative cells co - transfected with plasmids expressing Lyn - mCherry , MCP - GFPnls and 24xMS2 - k 5 3 ' UTRs . Right : percentage of cells with MCP - GFP localised to protrusions when co - transfected with full length or deletion versions of k 5 3 ' UTR ( n ≥ 3 experiments ) . white arrowheads indicate non - nuclear localisation of MCP - GFPnls ; arrows indicate the orientation of RNA localisation ; yellow dashed lines outline cell borders ; scale bars = 20 µm For each k 5 mRNA 3 ' UTR , a diagram of the full length 3 ' UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions ; black arrowheads indicate predicted polyadenylation

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

Figure 2E. Left: representative subconfluent motile cells co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-RAB13 3'UTR or 24xMS2. Right: percentage of cells with MCP-GFP localised to protrusions when co-transfected with full length or deletion versions of RAB13 3'UTR (n≥3 experiments). white arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate the orientation of RNA localisation; yellow dashed lines outline cell borders; scale bars = 20 µm For each k 5 mRNA 3'UTR, a diagram of the full length 3'UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions; black arrowheads indicate predicted polyadenylation sitesF. Left: representative cells co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-k 5 3'UTRs. Right: percentage of cells with MCP-GFP localised to protrusions when co-transfected with full length or deletion versions of k 5 3'UTR (n≥3 experiments). white arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate the orientation of RNA localisation; yellow dashed lines outline cell borders; scale bars = 20 µm For each k 5 mRNA 3'UTR, a diagram of the full length 3'UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions; black arrowheads indicate predicted polyadenylation sites


Figure 3B . Representative genotyping PCR demonstrates the band size shift in ∆ LE HUVECs . D . Wt and ∆ LE HUVEC RNAseq mapped reads depicting RAB13 exon usage . E . Quantification of RAB13 mRNA smFISH spot number in Wt and ∆ LE HUVECs ( n = 3 experiments ; ns : not significant ; unpaired t test ) . F . Number of RAB13 mRNA smFISH spots plotted against the respective Polarisation Index ( PI ) ( n = 29 cells ; ns : not significant ; linear regression ) . G . Left : representative Western blotting ( WB ) of Wt and ∆ LE HUVECs . Right : densitometry analysis of WB data ( n = 3 samples ; ns : not significant ; unpaired t test ) . H . smFISH co - detection of RAB13 and control GAPDH in Wt and ∆ LE motile HUVECs cultured under subconfluent conditions . Arrows indicate orientation of RNA localisation ; yellow dashed lines outline cell borders ; red circles highlight smFISH spots ; scale bars = 20 µm ( H ) . Bar charts are presented as means ± s . d . I . PI of RAB13 and GAPDH co - detected in Wt and ∆ LE HUVECs ( n = 29 cells ; P * * * < 0 . 001 , ns : not significant ; Mann - Whitney test ) . J . RAB13 PI plotted against respective GAPDH PI . The slope of the coloured lines represents the average RAB13 / GAPDH PI ratio ; the dashed grey line represents a 1 : 1 ratio ( n = 29 cells ) .

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

Figure 3B. Representative genotyping PCR demonstrates the band size shift in ∆LE HUVECs.D. Wt and ∆LE HUVEC RNAseq mapped reads depicting RAB13 exon usage.E. Quantification of RAB13 mRNA smFISH spot number in Wt and ∆LE HUVECs (n=3 experiments; ns: not significant; unpaired t test).F. Number of RAB13 mRNA smFISH spots plotted against the respective Polarisation Index (PI) (n=29 cells; ns: not significant; linear regression).G. Left: representative Western blotting (WB) of Wt and ∆LE HUVECs. Right: densitometry analysis of WB data (n=3 samples; ns: not significant; unpaired t test).H. smFISH co-detection of RAB13 and control GAPDH in Wt and ∆LE motile HUVECs cultured under subconfluent conditions. Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm (H). Bar charts are presented as means±s.d.I. PI of RAB13 and GAPDH co-detected in Wt and ∆LE HUVECs (n=29 cells; P***<0.001, ns: not significant; Mann-Whitney test).J. RAB13 PI plotted against respective GAPDH PI. The slope of the coloured lines represents the average RAB13/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n=29 cells).


Figure 4A . Representative time - lapse microscopy of a bEnd . 3 cell co - transfected with plasmids expressing Lyn - mCherry , MCP - GFPnls and 24xMS2 - RAB13 3 ' UTR . Arrowheads indicate filopodiaB . Frequency of newly formed filopodia formed within 5 µm intervals relative to the nearest MCP - GFPnls particle or a randomised ( ctrl ) position ( n = 99 filopodia ; * * P < 0 . 01 , ns : not significant ; Pearson r correlation ) . C . Distance of newly - formed filopodia to MCP - GFPnls or a ctrl position plotted against filopodia duration ( n = 99 filopodia ; * P < 0 . 05 , ns : not significant ; Spearman r correlation ) . D . Wt and ∆ LE HUVECs co - cultured on fibroblast monolayers . Endothelial cells were identified either with an antibody against the endothelial cell marker PECAM - 1 ( left ) or through expression of a nucleofected plasmid encoding the cytoskeletal marker Lifeact - GFP ( right ) . Arrowheads indicate filopodiaE . Number of filopodia detected in co - cultured HUVECs ( n = 30 cells ; * * P < 0 . 01 ; unpaired t test ) F . Number of filopodia detected in co - cultured HUVECs within 12 µm intervals relative to cell distal tip ( n = 30 cells ; * * P < 0 . 01 , P * * * < 0 . 001 , ns : not significant ; one - way ANOVA with Bonferroni ' s correction ) G . Number of filopodia detected in individual clones of co - cultured HUVECs within 12 µm intervals relative to cell distal tip .

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

Figure 4A. Representative time-lapse microscopy of a bEnd.3 cell co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-RAB13 3'UTR. Arrowheads indicate filopodiaB. Frequency of newly formed filopodia formed within 5 µm intervals relative to the nearest MCP-GFPnls particle or a randomised (ctrl) position (n=99 filopodia; **P<0.01, ns: not significant; Pearson r correlation).C. Distance of newly-formed filopodia to MCP-GFPnls or a ctrl position plotted against filopodia duration (n=99 filopodia; *P<0.05, ns: not significant; Spearman r correlation).D. Wt and ∆LE HUVECs co-cultured on fibroblast monolayers. Endothelial cells were identified either with an antibody against the endothelial cell marker PECAM-1 (left) or through expression of a nucleofected plasmid encoding the cytoskeletal marker Lifeact-GFP (right). Arrowheads indicate filopodiaE. Number of filopodia detected in co-cultured HUVECs (n=30 cells; **P<0.01; unpaired t test)F. Number of filopodia detected in co-cultured HUVECs within 12 µm intervals relative to cell distal tip (n=30 cells; **P<0.01, P***<0.001, ns: not significant; one-way ANOVA with Bonferroni's correction)G. Number of filopodia detected in individual clones of co-cultured HUVECs within 12 µm intervals relative to cell distal tip.


Figure 5B . Representative Puro - PLA experiments detecting newly synthesised RAB13 in HUVEC protrusions present in the lower side of Transwell membranes . Puro : puromycin ; Aniso : anisomycin ; 1ary Abs : primary antibodies . white arrowheads indicate Puro - PLA punctae ; yellow dashed lines outline protrusion borders ( B ) ; C . Quantification of RAB13 Puro - PLA punctae normalised to protrusion area ( n ≥ 40 protrusions ; * P < 0 . 05 , * * * * P < 0 . 0001 ; Kruskal - Wallis test with Dunn ' s correction ) . D . Representative RAB13 IF assay on migrating HUVECs . E . Left : representative Western blotting ( WB ) of siRNA transfected HUVECs . Right : densitometry analysis of WB data ( n = 3 samples ; * P < 0 . 05 ; paired t test ) . F . Control ( ctrl ) and RAB13 siRNA - treated HUVECs co - cultured on fibroblast monolayers . Endothelial cells were identified with an antibody against the endothelial cell marker PECAM - 1 . black arrowheads indicate filopodia ( F ) G . Number of filopodia detected in co - cultured HUVECs ( n ≥ 35 cells ; * * P < 0 . 01 ; unpaired t test ) . H . Number of filopodia detected in co - cultured HUVECs within 12 µm intervals relative to cell distal tip ( n ≥ 35 cells ; * P < 0 . 05 , ns : not significant ; Kruskal - Wallis test with Dunn ' s correction ) .

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

Figure 5B. Representative Puro-PLA experiments detecting newly synthesised RAB13 in HUVEC protrusions present in the lower side of Transwell membranes. Puro: puromycin; Aniso: anisomycin; 1ary Abs: primary antibodies. white arrowheads indicate Puro-PLA punctae; yellow dashed lines outline protrusion borders (B);C. Quantification of RAB13 Puro-PLA punctae normalised to protrusion area (n≥40 protrusions; *P<0.05, ****P<0.0001; Kruskal-Wallis test with Dunn's correction).D. Representative RAB13 IF assay on migrating HUVECs.E. Left: representative Western blotting (WB) of siRNA transfected HUVECs. Right: densitometry analysis of WB data (n=3 samples; *P<0.05; paired t test).F. Control (ctrl) and RAB13 siRNA-treated HUVECs co-cultured on fibroblast monolayers. Endothelial cells were identified with an antibody against the endothelial cell marker PECAM-1. black arrowheads indicate filopodia (F)G. Number of filopodia detected in co-cultured HUVECs (n≥35 cells; **P<0.01; unpaired t test).H. Number of filopodia detected in co-cultured HUVECs within 12 µm intervals relative to cell distal tip (n≥35 cells; *P<0.05, ns: not significant; Kruskal-Wallis test with Dunn's correction).


Figure 6A . Left : Tg ( fli1ep : MCP - GFPnls ) zebrafish embryo at 26 hours post fertilisation ( hpf ) displaying vascular - specific expression of MCP - GFPnls . Inset shows the nuclear expression of MCP - GFPnls in the intersomitic vessels ( ISVs ) sprouting from the dorsal aorta ( DA ) . Middle : scheme depicts the in vivo MS2 system strategy with fli1 enhancer / promoter ( fli1ep ) - driven expression of reporter constructs , simultaneous translation of Lyn - mCherry reporter and binding of MCP - GFPnls to 24xMS2 - rab13 3 ' UTR . Right : scheme illustrates ISV cells expressing Lyn - mCherry imaged in panels B - D . TC : tip cell ; SC : stalk cell . Arrowheads indicate non - nuclear localisation of MCP - GFPnls ; arrows indicate direction of ISV sprouting ; yellow dashed lines outline ISV ( A ) ( B - D ) Time - lapse microscopy of Tg ( fli1ep : MCP - GFPnls ) tip ( C ) and stalk ( B , D ) cells displaying mosaic expression of Lyn - mCherry - 24xMS2 - rab13 3 ' UTR in ISV cells . Arrowheads indicate non - nuclear localisation of MCP - GFPnls ; arrows indicate direction of ISV sprouting ; yellow dashed lines outline ISV cell ( B - D )

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

Figure 6A. Left: Tg(fli1ep:MCP-GFPnls) zebrafish embryo at 26 hours post fertilisation (hpf) displaying vascular-specific expression of MCP-GFPnls. Inset shows the nuclear expression of MCP-GFPnls in the intersomitic vessels (ISVs) sprouting from the dorsal aorta (DA). Middle: scheme depicts the in vivo MS2 system strategy with fli1 enhancer/promoter (fli1ep)-driven expression of reporter constructs, simultaneous translation of Lyn-mCherry reporter and binding of MCP-GFPnls to 24xMS2-rab13 3'UTR. Right: scheme illustrates ISV cells expressing Lyn-mCherry imaged in panels B-D. TC: tip cell; SC: stalk cell. Arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate direction of ISV sprouting; yellow dashed lines outline ISV (A)(B-D) Time-lapse microscopy of Tg(fli1ep:MCP-GFPnls) tip (C) and stalk (B,D) cells displaying mosaic expression of Lyn-mCherry-24xMS2-rab13 3'UTR in ISV cells. Arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate direction of ISV sprouting; yellow dashed lines outline ISV cell (B-D) borders


Figure 7B . Representative genotyping PCR demonstrates the band size shift in zebrafish harbouring a ∆ 482 rab13 3 ' UTR . Asterisk marks a heteroduplex formed between Wt and ∆ 482 rab13 3 ' UTR PCR amplicons . D . RNAseq mapped reads depicting rab13 exon usage in Tg ( kdrl : EGFP ) rab13 + / + and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR zebrafish embryos . Coloured lines indicate SNPs . E . qPCR analysis of rab13 mRNA levels in individual 26 - 28 hpf clutch - matched sibling embryos ( n ≥ 9 embryos ; ns : not significant ; Kruskal - Wallis test with Dunn ' s correction ) . : + / + , + / ∆ and ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR embryos , respectivelyF . smFISH detection of rab13 and kdr mRNA in cultured GFP - expressing endothelial cells extracted from 48 hpf Tg ( kdrl : EGFP ) rab13 + / + and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR zebrafish embryos . Arrows indicate orientation of RNA localisation ; yellow dashed lines outline cell borders ; red circles highlight smFISH spots ; scale bars = 10 µm ( F ) . G . Polarisation Index ( PI ) of rab13 and kdr detected by smFISH in individual zebrafish cells ( n ≥ 8 cells ; * * P < 0 . 01 , ns : not significant ; one - way ANOVA with Bonferroni ' s correction ) . : + / + , + / ∆ and ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR embryos , respectivelyH . rab13 PI plotted against respective kdr PI . The slope of the coloured lines represents the average rab13 / kdr PI ratio ; the dashed grey line represents a 1 : 1 ratio ( n ≥ 8 cells ) . : + / + , + / ∆ and ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR embryos ,

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

Figure 7B. Representative genotyping PCR demonstrates the band size shift in zebrafish harbouring a ∆482 rab13 3'UTR. Asterisk marks a heteroduplex formed between Wt and ∆482 rab13 3'UTR PCR amplicons.D. RNAseq mapped reads depicting rab13 exon usage in Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR zebrafish embryos. Coloured lines indicate SNPs.E. qPCR analysis of rab13 mRNA levels in individual 26-28 hpf clutch-matched sibling embryos (n≥9 embryos; ns: not significant; Kruskal-Wallis test with Dunn's correction). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectivelyF. smFISH detection of rab13 and kdr mRNA in cultured GFP-expressing endothelial cells extracted from 48 hpf Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR zebrafish embryos. Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 10 µm (F).G. Polarisation Index (PI) of rab13 and kdr detected by smFISH in individual zebrafish cells (n≥8 cells; **P<0.01, ns: not significant; one-way ANOVA with Bonferroni's correction). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectivelyH. rab13 PI plotted against respective kdr PI. The slope of the coloured lines represents the average rab13/kdr PI ratio; the dashed grey line represents a 1:1 ratio (n≥8 cells). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively


Figure 8A . Time - lapse confocal microscopy of representative Tg ( kdrl : EGFP ) rab13 + / + and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR embryos . DLAV : dorsal longitudinal anastomotic vessel ; HM : horizontal myoseptum . T0 = 25 hpf ; arrowheads indicate extra branches emerging from the main ISVs at the HM position ; scale bars = 50 µm ( A ) . B . Frequency of ISV ectopic branching occurring at the HM ( n = 4 experiments ; * P < 0 . 05 ; one - way ANOVA with Bonferroni ' s correction ) . + / + , + / ∆ and ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR embryos , respectively ( B ) .

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

Figure 8A. Time-lapse confocal microscopy of representative Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR embryos. DLAV: dorsal longitudinal anastomotic vessel; HM: horizontal myoseptum. T0= 25 hpf; arrowheads indicate extra branches emerging from the main ISVs at the HM position; scale bars = 50 µm (A).B. Frequency of ISV ectopic branching occurring at the HM (n=4 experiments; *P<0.05; one-way ANOVA with Bonferroni's correction). +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively (B).


figf1 ( a - d ) Rotenone ( Rot ) increased the number of GFP - LC3 puncta and the level of co - localization with mitochondria ( arrows ) in SH - SY5Y cells ( a - c ; 1 μM ) and primary cortical neurons ( d ; 250 nM ) , quantified in Fig . 3f and Supplementary Fig . S1b , c . Veh , vehicle . ( e , f ) Rotenone increased delivery of MitoTracker Green - stained mitochondria ( mt ) to LysoTracker Red ( LTR ) - stained lysosomes , inhibited by siRNA knockdown of ATG7 or LC3 in SH - SY5Y cells . Ctrl , control . Right : RNAi knockdown . ( g ) Rotenone decreased IMM ( COXIV ) , OMM ( TOM40 ) and matrix ( MnSOD ) protein expression levels in primary neurons , reversed by bafilomycin ( Baf ) inhibition of autolysosomal degradation , quantified in Supplementary Fig . S1e . Mean ± s . d . of n = 7 independent experiments for b , c , and n = 3 independent experiments for f , g ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle control ; † P0 . 05 versus Rot / control siRNA . Scale bars , 10 μm . See Supplementary Fig . S7 for uncropped blots .

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

figf1(a-d) Rotenone (Rot) increased the number of GFP-LC3 puncta and the level of co-localization with mitochondria (arrows) in SH-SY5Y cells (a-c; 1 μM) and primary cortical neurons (d; 250 nM), quantified in Fig. 3f and Supplementary Fig. S1b,c. Veh, vehicle.(e,f) Rotenone increased delivery of MitoTracker Green-stained mitochondria (mt) to LysoTracker Red (LTR)-stained lysosomes, inhibited by siRNA knockdown of ATG7 or LC3 in SH-SY5Y cells. Ctrl, control. Right: RNAi knockdown.(g) Rotenone decreased IMM (COXIV), OMM (TOM40) and matrix (MnSOD) protein expression levels in primary neurons, reversed by bafilomycin (Baf) inhibition of autolysosomal degradation, quantified in Supplementary Fig. S1e. Mean ± s.d. of n = 7 independent experiments for b,c, and n = 3 independent experiments for f,g (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control; †P0.05 versus Rot/control siRNA. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.


figf2 ( a , b ) IMM and OMM fractions isolated from primary neurons following the indicated treatments were lipid extracted for LC - MS analysis . ( a ) MS spectra demonstrated increased cardiolipin ( CL ) content of the OMM after rotenone ( Rot ) treatment , with diversification of the cluster distribution to the 7 clusters exhibited by the IMM . Veh , vehicle . ( b ) Pie charts showing the CL distribution between IMM and OMM fractions from toxin - treated neurons , normalized to mitochondrial lipid phosphorus . ( c , d ) Treatment with rotenone caused significant increases in PLA2 - hydrolysable ( surface accessible ) CL assessed by LC - MS in primary cortical neurons ( c ; 250 nM ) and SH - SY5Y cells ( d ; 1 μM ) . ( e , f ) Treatment with 6 - OHDA and CCCP increased surface exposure of CL probed with annexin V in SH - SY5Y ( e , 120 μM ) and parkin - expressing HeLa cells ( f , 20 μM ) , respectively . Mean ± s . d . of n = 3 independent experiments for c - f ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle control .

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

figf2(a,b) IMM and OMM fractions isolated from primary neurons following the indicated treatments were lipid extracted for LC-MS analysis. (a) MS spectra demonstrated increased cardiolipin (CL) content of the OMM after rotenone (Rot) treatment, with diversification of the cluster distribution to the 7 clusters exhibited by the IMM. Veh, vehicle. (b) Pie charts showing the CL distribution between IMM and OMM fractions from toxin-treated neurons, normalized to mitochondrial lipid phosphorus.(c,d) Treatment with rotenone caused significant increases in PLA2-hydrolysable (surface accessible) CL assessed by LC-MS in primary cortical neurons (c; 250 nM) and SH-SY5Y cells (d; 1 μM).(e,f) Treatment with 6-OHDA and CCCP increased surface exposure of CL probed with annexin V in SH-SY5Y (e, 120 μM) and parkin-expressing HeLa cells (f, 20 μM), respectively. Mean ± s.d. of n = 3 independent experiments for c-f (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control.


figf3 ( a - e ) SH - SY5Y cells expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , and treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( a ) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647 - annexin V binding . ( a - e ) SH - SY5Y cells expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , and treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( b - d ) PLS3 knockdown had no effect on rotenone - induced autophagy ( b ) , but decreased mitophagy in SH - SY5Y cells treated with rotenone ( c ; 1 μM ) or 6 - OHDA ( d ; 120 μM ) . ( a - e ) SH - SY5Y cells expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , and treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( d , e ) The effects of PLS3 siRNA were recapitulated using a second siRNA # 433 ( d , Supplementary Fig . S3b , g ) , and reversed by transfection with an RNAi - resistant mouse PLS3 vector ( e ) . Inset : PLS3 overexpression . ( f - i ) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone ( 250 nM ) and analysed for autophagy ( Supplementary Fig . S1b ) or mitophagy by either co - localization analysis ( f ) ( f - i ) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone ( 250 nM ) and analysed for autophagy ( Supplementary Fig . S1b ) or mitophagy by either co - localization analysis ( f ) , or with mitochondrial protein immunoblotting ( g ) and densitometry ( h , i and Supplementary Fig . S4c ) . Knockdown efficiencies are shown in Supplementary Figs S3 and S4 . Mean ± s . d . of n = 4 independent experiments for b , c , and n = 3 independent experiments for a , d - f , h - i ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle ; † P0 . 05 versus toxin - treated control siRNA ; * * P0 . 05 versus Rot / PLS3 siRNA . See Supplementary Fig . S7 for uncropped blots .

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

figf3(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (a) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647-annexin V binding.(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (b-d) PLS3 knockdown had no effect on rotenone-induced autophagy (b), but decreased mitophagy in SH-SY5Y cells treated with rotenone (c; 1 μM) or 6-OHDA (d; 120 μM).(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (d,e) The effects of PLS3 siRNA were recapitulated using a second siRNA #433 (d, Supplementary Fig. S3b,g), and reversed by transfection with an RNAi-resistant mouse PLS3 vector (e). Inset: PLS3 overexpression.(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f)(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f), or with mitochondrial protein immunoblotting (g) and densitometry (h,i and Supplementary Fig. S4c). Knockdown efficiencies are shown in Supplementary Figs S3 and S4. Mean ± s.d. of n = 4 independent experiments for b,c, and n = 3 independent experiments for a,d-f,h-i (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle; †P0.05 versus toxin-treated control siRNA; **P0.05 versus Rot/PLS3 siRNA. See Supplementary Fig. S7 for uncropped blots.


figf4 ( a ) Western blot analysis showed beclin 1 cleavage products ( Mr49 K ) were not observed in primary neurons exposed to mitophagy - inducing sublethal concentrations of rotenone ( 250 nM × 2 h : Rot low ) or STS ( 100 nM × 2 h ) . However , beclin 1 was cleaved on exposure to a lethal dose of rotenone ( 1 mM × 24 h : Rot high ) . Veh , vehicle ; CTD , C - terminal domain . ( b , c ) Likewise , SH - SY5Y cells did not show beclin 1 cleavage on exposure to rotenone ( b ) or 6 - OHDA ( c ; duplicate lanes ) . Data are representative of 2 - 3 independent experiments per toxin . ( d ) Cell lysates from stable PINK1 - Flag - expressing SH - SY5Y cells were treated with 6 - OHDA ( 120 μM ) , rotenone ( 1 μM ) or FCCP ( 2 μM ) , and analysed by Flag immunoblot for PINK1 - Flag levels . ( e ) SH - SY5Y cells transfected with HA - parkin were treated with the indicated toxins , immunolabelled for HA ( green ) and mitochondrial p60 antigen ( red ) and analysed for HA - parkin redistribution to mitochondria . FCCP elicited translocation of parkin to mitochondria ( arrows ) , which was not seen with the other mitophagy stimuli . ( f ) Cytosolic and mitochondrial pellets were prepared from SH - SY5Y cells treated with vehicle ( V ) , 6 - OHDA ( O , 120 μM ) or rotenone ( R , 1 μM ) . Gels were loaded with cytosolic or mitochondrial proteins , and immunoblotted for p62 / SQSMT1 , and the indicated fractionation markers . ( g , h ) Confocal analysis of p62 / SQSMT1 distribution in SH - SY5Y cells ( g ) and in primary cortical neurons ( h ) that were co - transfected with mCherry - tagged p62 / SQSMT1 and mitochondrially targeted GFP . Note recruitment of p62 to large mitochondrial aggregates ( g , yellow , arrows ) in FCCP - treated cells , which was not observed in control or rotenone - treated cells . Scale bars , 10 μm . See Supplementary Fig . S7 for uncropped blots .

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

figf4(a) Western blot analysis showed beclin 1 cleavage products (Mr49 K) were not observed in primary neurons exposed to mitophagy-inducing sublethal concentrations of rotenone (250 nM×2 h: Rot low) or STS (100 nM×2 h). However, beclin 1 was cleaved on exposure to a lethal dose of rotenone (1 mM×24 h: Rot high). Veh, vehicle; CTD, C-terminal domain.(b,c) Likewise, SH-SY5Y cells did not show beclin 1 cleavage on exposure to rotenone (b) or 6-OHDA (c; duplicate lanes). Data are representative of 2-3 independent experiments per toxin.(d) Cell lysates from stable PINK1-Flag-expressing SH-SY5Y cells were treated with 6-OHDA (120 μM), rotenone (1 μM) or FCCP (2 μM), and analysed by Flag immunoblot for PINK1-Flag levels.(e) SH-SY5Y cells transfected with HA-parkin were treated with the indicated toxins, immunolabelled for HA (green) and mitochondrial p60 antigen (red) and analysed for HA-parkin redistribution to mitochondria. FCCP elicited translocation of parkin to mitochondria (arrows), which was not seen with the other mitophagy stimuli.(f) Cytosolic and mitochondrial pellets were prepared from SH-SY5Y cells treated with vehicle (V), 6-OHDA (O, 120 μM) or rotenone (R, 1 μM). Gels were loaded with cytosolic or mitochondrial proteins, and immunoblotted for p62/SQSMT1, and the indicated fractionation markers.(g,h) Confocal analysis of p62/SQSMT1 distribution in SH-SY5Y cells (g) and in primary cortical neurons (h) that were co-transfected with mCherry-tagged p62/SQSMT1 and mitochondrially targeted GFP. Note recruitment of p62 to large mitochondrial aggregates (g, yellow, arrows) in FCCP-treated cells, which was not observed in control or rotenone-treated cells. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.