DNA repair of MMS damage . Chromosome VII species revealed by hybridization with ADE5 , 7 probes in pulsed ‐ field gel electrophoresis ( PFGE ) . Asynchronously growing WT and rpb1 cells were exposed to 0 . 1 % MMS for 30 min , washed to remove MMS and cultured in MMS ‐ free medium for the indicated periods to allow repair . Repair was calculated as the percentage of FLC appearing after MMS treatment . The FLC signal was quantified with respect to the total signal of each lane . Data show the mean and SD of 3 independent experiments . NLC , nonlinear chromosome . FLC , full ‐ length linear chromosome . CF , chromosome fragment resulting from DNA breaks .

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DNA repair of MMS damage. Chromosome VII species revealed by hybridization with ADE5,7 probes in pulsed‐field gel electrophoresis (PFGE). Asynchronously growing WT and rpb1 cells were exposed to 0.1% MMS for 30 min, washed to remove MMS and cultured in MMS‐free medium for the indicated periods to allow repair. Repair was calculated as the percentage of FLC appearing after MMS treatment. The FLC signal was quantified with respect to the total signal of each lane. Data show the mean and SD of 3 independent experiments. NLC, nonlinear chromosome. FLC, full‐length linear chromosome. CF, chromosome fragment resulting from DNA breaks.


Retention of RNAPII in the actively transcribed PMA1 gene in RNAPII mutants . ChIP analysis of RNAPII in asynchronously growing WT , rpb1 ‐ 1 , rpb1 ‐ S751F , rpb2 ‐ 10 and rpb9Δ cells at the PMA1 gene . The scheme of the gene and the PCR ‐ amplified fragments are shown . Recruitment values were calculated from the amount of DNA present in each region , normalizing IP / input ratios , and relative to the amount of DNA of the intergenic region . Error bars indicate the standard deviation ( SD ) .

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Retention of RNAPII in the actively transcribed PMA1 gene in RNAPII mutants. ChIP analysis of RNAPII in asynchronously growing WT, rpb1‐1, rpb1‐S751F, rpb2‐10 and rpb9Δ cells at the PMA1 gene. The scheme of the gene and the PCR‐amplified fragments are shown. Recruitment values were calculated from the amount of DNA present in each region, normalizing IP/input ratios, and relative to the amount of DNA of the intergenic region. Error bars indicate the standard deviation (SD).


Northern blot analysis of the expression of PMA1 gene . RNA was isolated from mid ‐ log phase cultures grown in YPD ‐ rich medium . As 32P ‐ labeled DNA probes , an internal 776 ‐ bp PMA1 fragment and an internal 513 ‐ bp SCR1 fragment obtained by PCR were used . Bars indicate the SD of 2 independent experiments .

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Northern blot analysis of the expression of PMA1 gene. RNA was isolated from mid‐log phase cultures grown in YPD‐rich medium. As 32P‐labeled DNA probes, an internal 776‐bp PMA1 fragment and an internal 513‐bp SCR1 fragment obtained by PCR were used. Bars indicate the SD of 2 independent experiments.


Analysis of Rrm3 recruitment in WT and rpb1 ‐ 1 . Rrm3 IP histogram bars in the y ‐ axis show the average signal log2 ratio of loci enriched in the immunoprecipitated fraction along the indicated regions in the x ‐ axis . When the ratio fulfills the P ‐ value criteria ( P 0 . 01 ) it is shown in red .

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Analysis of Rrm3 recruitment in WT and rpb1‐1. Rrm3 IP histogram bars in the y‐axis show the average signal log2 ratio of loci enriched in the immunoprecipitated fraction along the indicated regions in the x‐axis. When the ratio fulfills the P‐value criteria (P 0.01) it is shown in red.


Composite profile of Rrm3 occupancy across the average ORF plotted as Rrm3 percentage of significant ChIP hits per segment . Each gene was divided into 10 equivalent segments from the start and end coordinates ( segments 2 - 10 ) . Two additional segments of the same size were considered upstream ( 5 ′ ) and downstream ( 3 ′ ) of each ORF ( segments 1 and 12 , respectively ) . The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted .

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Composite profile of Rrm3 occupancy across the average ORF plotted as Rrm3 percentage of significant ChIP hits per segment. Each gene was divided into 10 equivalent segments from the start and end coordinates (segments 2-10). Two additional segments of the same size were considered upstream (5′) and downstream (3′) of each ORF (segments 1 and 12, respectively). The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted.


Composite profile of Rrm3 occupancy across the average ARS plotted as Rrm3 percentage of significant ChIP hits per segment . Each ARS was divided into 11 equivalent segments from the start and end coordinates . The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted .

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Composite profile of Rrm3 occupancy across the average ARS plotted as Rrm3 percentage of significant ChIP hits per segment. Each ARS was divided into 11 equivalent segments from the start and end coordinates. The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted.


( C ) PCR analysis of a pool of transfected cells after treatment with Cas9 - D10A ' nickase ' and the indicated combination of gRNAs The ~ 200 bp product was centred around the start of exon 1 of the SLD5 gene . Efficient cutting of both template strands by Cas9 - nickase was indicated by the production of a smeary PCR product ( gRNAs 2 + 3 ) , reflecting DNA repair that mostly produced small deletions around the nicking site The combination of gRNAs 1 + 3 did not produce a smeary PCR , due to a polymorphism within the PAM motif of the genomic

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(C) PCR analysis of a pool of transfected cells after treatment with Cas9-D10A 'nickase' and the indicated combination of gRNAs The ~200 bp product was centred around the start of exon 1 of the SLD5 gene. Efficient cutting of both template strands by Cas9-nickase was indicated by the production of a smeary PCR product (gRNAs 2+3), reflecting DNA repair that mostly produced small deletions around the nicking site The combination of gRNAs 1+3 did not produce a smeary PCR, due to a polymorphism within the PAM motif of the genomic locus


( F ) Immunoblot indicating successful tagging of both alleles of SLD5 with TAP or GFP .

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(F) Immunoblot indicating successful tagging of both alleles of SLD5 with TAP or GFP.


( G ) Asynchronous cultures of the indicated cells were used to generate cell extracts , which were then incubated with agarose beads coupled to anti - GFP nanobodies .

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(G) Asynchronous cultures of the indicated cells were used to generate cell extracts, which were then incubated with agarose beads coupled to anti-GFP nanobodies.


( H ) Extracts of TAP - SLD5 mouse ES cells were incubated plus or minus DNase for 30 minutes at 4 ° C , before immunoprecipitation of TAP - SLD5 .

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(H) Extracts of TAP-SLD5 mouse ES cells were incubated plus or minus DNase for 30 minutes at 4 °C, before immunoprecipitation of TAP-SLD5.


( I ) TAP - SLD5 cells were synchronised as indicated DNA content was monitored throughout the experiment by flow cytometry .

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(I) TAP-SLD5 cells were synchronised as indicated DNA content was monitored throughout the experiment by flow cytometry.


Samples were taken at the indicated times and used to isolate TAP - SLD5 by immunoprecipitation on IgG beads . The indicated proteins were monitored by immunoblotting . For reasons of space , samples A , S , M and 1 - 6 were resolved in a separate gel to samples 7 - 8 .

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Samples were taken at the indicated times and used to isolate TAP-SLD5 by immunoprecipitation on IgG beads. The indicated proteins were monitored by immunoblotting. For reasons of space, samples A, S, M and 1-6 were resolved in a separate gel to samples 7-8.


( A ) Cells were treated with 5 µM CB - 5083 as indicated ( p97i = p97 inhibitor ) . Extracts were then incubated with IgG beads to isolate the GINS complex via TAP - SLD5 and the indicated factors were monitored by immunoblotting . Accumulation of the CMG helicase upon inhibition of p97 was reflected by the increased association of GINS with CDC45 and MCM2 - 7 .

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(A) Cells were treated with 5 µM CB-5083 as indicated (p97i = p97 inhibitor). Extracts were then incubated with IgG beads to isolate the GINS complex via TAP-SLD5 and the indicated factors were monitored by immunoblotting. Accumulation of the CMG helicase upon inhibition of p97 was reflected by the increased association of GINS with CDC45 and MCM2-7.


( B ) Immunostaining of HP1 protein in fixed mouse ES cells that were stained with Hoechst to reveal condensed patches of heterochromatic DNA ( examples marked by arrows ; colocalization of HP1 and condensed DNA in 83 % of cells , n = 103 ) .

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(B) Immunostaining of HP1 protein in fixed mouse ES cells that were stained with Hoechst to reveal condensed patches of heterochromatic DNA (examples marked by arrows; colocalization of HP1 and condensed DNA in 83 % of cells, n = 103).


( C ) Time - lapse analysis of mouse ES cells expressing GFP - SLD5 and mCherry - PCNA Arrows indicate co - localisation on heterochromatic patches , which was observed in 14 % asynchronous cells ( n = 238 ) . GFP - SLD5 appeared and disappeared with similar kinetics to mCherry - PCNA ( 100 % cells , n = 101 ) .

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(C) Time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-PCNA Arrows indicate co-localisation on heterochromatic patches, which was observed in 14 % asynchronous cells (n = 238). GFP-SLD5 appeared and disappeared with similar kinetics to mCherry-PCNA (100 % cells, n = 101).


( D ) GFP - SLD5 mouse ES cells were treated with p97i as indicated and then fixed and stained with Hoechst . Arrows indicate accumulation of GFP - SLD5 on constitutive heterochromatin ( 53 % cells , n = 125 ) .

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(D) GFP-SLD5 mouse ES cells were treated with p97i as indicated and then fixed and stained with Hoechst. Arrows indicate accumulation of GFP-SLD5 on constitutive heterochromatin (53 % cells, n = 125).


( E ) Immunofluorescence of the replisome components CLASPIN ( i ) and CTF4 ( ii ) in GFP - SLD5 cells treated with p97i . Arrows indicate co - localisation of GFP - SLD5 patches with CLASPIN ( 95 % cells , n = 107 ) or CTF4 ( 91 % cells , n = 45 ) . Scale bars in all panels correspond to 10 µm .

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(E) Immunofluorescence of the replisome components CLASPIN (i) and CTF4 (ii) in GFP-SLD5 cells treated with p97i. Arrows indicate co-localisation of GFP-SLD5 patches with CLASPIN (95 % cells, n = 107) or CTF4 (91 % cells, n = 45). Scale bars in all panels correspond to 10 µm.


( A ) Cell extracts were generated after the indicated treatments and then incubated with anti - GFP beads . Factors co - purifying with GFP - SLD5 were monitored by immunoblotting .

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(A) Cell extracts were generated after the indicated treatments and then incubated with anti-GFP beads. Factors co-purifying with GFP-SLD5 were monitored by immunoblotting.


( B ) TAP - SLD5 cells were subjected to the indicated treatments . The level of LRR1 and VHL was assessed by immunoblotting . The asterisk denotes a non - specific band . ( C ) For the experiment described in ( B ) , TAP - SLD5 was isolated from cell extracts and the indicated factors monitored by immunoblotting .

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(B) TAP-SLD5 cells were subjected to the indicated treatments. The level of LRR1 and VHL was assessed by immunoblotting. The asterisk denotes a non-specific band. (C) For the experiment described in (B), TAP-SLD5 was isolated from cell extracts and the indicated factors monitored by immunoblotting.


( D ) The activity of CUL2LRR1 was inhibited in GFP - SLD5 cells , by treatment with a combination of LRR1 siRNA and MLN4924 Fixed cells were analysed Arrows indicate the accumulation of GFP - SLD5 on constitutive heterochromatin ( 49 % cells , n = 125 ) . ( E ) Cells were treated as in ( D ) and the localisation of GFP - SLD5 was compared with CLASPIN by immunofluorescence . Arrows indicate the co - localisation of GFP - SLD5 and CLASPIN on constitutive heterochromatin ( in 90 % cells with GFP - SLD5 patches , n = 103 ) .

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(D) The activity of CUL2LRR1 was inhibited in GFP-SLD5 cells , by treatment with a combination of LRR1 siRNA and MLN4924 Fixed cells were analysed Arrows indicate the accumulation of GFP-SLD5 on constitutive heterochromatin (49 % cells, n = 125). (E) Cells were treated as in (D) and the localisation of GFP-SLD5 was compared with CLASPIN by immunofluorescence. Arrows indicate the co-localisation of GFP-SLD5 and CLASPIN on constitutive heterochromatin (in 90 % cells with GFP-SLD5 patches, n = 103).


( A ) Time - lapse analysis of mouse ES cells expressing GFP - SLD5 and mCherry - PCNA , following inhibition of CUL2LRR1 . Arrows illustrate the persistence of GFP - SLD5 on heterochromatin after disappearance of mCherry - PCNA .

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(A) Time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-PCNA, following inhibition of CUL2LRR1. Arrows illustrate the persistence of GFP-SLD5 on heterochromatin after disappearance of mCherry-PCNA.


( B ) time - lapse analysis of mouse ES cells expressing GFP - SLD5 and mCherry - Histone H2B ( from the CAG promoter at the ROSA26 locus after inhibition of CUL2LRR1 . Arrows indicate heterochromatic patches of GFP - SLD5 that disappear during entry into mitosis ( 98 % cells , n = 97 ) . ( C ) Equivalent data to ( B ) but for untreated cells ( 100 % of untreated cells lacked GFP - SLD5 on mitotic chromatin , n = 149 ) .

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(B) time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-Histone H2B (from the CAG promoter at the ROSA26 locus after inhibition of CUL2LRR1. Arrows indicate heterochromatic patches of GFP-SLD5 that disappear during entry into mitosis (98 % cells, n = 97). (C) Equivalent data to (B) but for untreated cells (100 % of untreated cells lacked GFP-SLD5 on mitotic chromatin, n = 149).


( A ) TRAIP co - purifies with the CMG helicase from extracts of mouse ES cells . The asterisk indicates a non - specific band .

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(A) TRAIP co-purifies with the CMG helicase from extracts of mouse ES cells. The asterisk indicates a non-specific band.


( B ) Mouse ES cells were processed Arrows in ( i ) indicate accumulation of GFP - SLD5 on heterochromatin during interphase , whereas arrows in ( ii ) denote GFP - SLD5 on chromatin during mitotic metaphase . Scale bars correspond to 10 µm .

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(B) Mouse ES cells were processed Arrows in (i) indicate accumulation of GFP-SLD5 on heterochromatin during interphase, whereas arrows in (ii) denote GFP-SLD5 on chromatin during mitotic metaphase. Scale bars correspond to 10 µm.


( C ) Immunoblot illustrating the stable expression from the CAG promoter of the indicated forms of TRAIP at the ROSA26 locus in TRAIP - / - mouse ES cells . Asterisks indicate non - specific bands . ( D ) Quantification of the percentage of mitotic cells with GFP - SLD5 on mitotic chromosomes , after inhibition of CUL2LRR1 in cells with the indicated genotypes . Cells were fixed before analysis .

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(C) Immunoblot illustrating the stable expression from the CAG promoter of the indicated forms of TRAIP at the ROSA26 locus in TRAIP -/- mouse ES cells. Asterisks indicate non-specific bands. (D) Quantification of the percentage of mitotic cells with GFP-SLD5 on mitotic chromosomes, after inhibition of CUL2LRR1 in cells with the indicated genotypes. Cells were fixed before analysis.


B 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 .

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B 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 .

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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 .

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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 .

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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 .

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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.


A 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 ) .

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

A 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 ) .

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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 .

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

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 .

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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 .

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

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 .

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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 .

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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 .

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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.


A 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 .

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

A 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 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 .

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 .

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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 .

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 .

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

F 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 .

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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.


A 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 .

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

A 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 .

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

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

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

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.


A : 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 ) .

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

A: 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 .

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

C: 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.


A , 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 .

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

A, 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 .

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

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 .

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

E: 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 .

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

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 .

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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 .

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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.


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 ) .

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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 .

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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 ) .

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 .

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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 .

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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 ) .

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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

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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


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

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.


A - 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 .

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A-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 .

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 .

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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 .

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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 .

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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.


A : 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 .

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A: 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 .

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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 .

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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 .

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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.


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 .

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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 ) .

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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 ) .

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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 .

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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.


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 .

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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.


B . 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 .

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B. 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 .

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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

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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 µm


E . 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 ) .

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E. 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 ) .

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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

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 µm


H . 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 ) .

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H. 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 ) .

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I. PIs of k 2 mRNAs and GAPDH co-detected in HUVECs (n≥19 cells; *P<0.05; Wilcoxon test).


E . 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

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

E. 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 sites


F . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. 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


B . Representative genotyping PCR demonstrates the band size shift in ∆ LE HUVECs .

B. Representative genotyping PCR demonstrates the band size shift in ∆LE HUVECs.


D . Wt and ∆ LE HUVEC RNAseq mapped reads depicting RAB13 exon usage .

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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 ) .

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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 ) .

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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 ) .

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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 .

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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 ) .

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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 ) .

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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).


A . 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

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A. 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 filopodia


B . 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 ) .

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B. 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 ) .

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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

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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 filopodia


E . Number of filopodia detected in co - cultured HUVECs ( n = 30 cells ; * * P < 0 . 01 ; unpaired t test )

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E. 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 )

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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 .

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G. Number of filopodia detected in individual clones of co-cultured HUVECs within 12 µm intervals relative to cell distal tip.


B . 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 ) ;

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B. 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 ) .

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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 .

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 ) .

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 )

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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 ) .

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

H. 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).


A . 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 )

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A. 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 )

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(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


B . 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 .

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B. 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 .

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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 ,

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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, respectively