( A Bulk - seq of DE genes ( DEG ) significantly suppressed ( FDR < 0 . 05 ; generalized linear model extraction ( GLM ) by edgeR ) in proliferating RM1 bone - derived ( BD ) compared to parental RM1 lung metastases . - intrinsic type I IFN signaling is suppressed in proliferating bone lesions ( n = 1 ) compared to parental RM1 ( n = 2 ) as shown by ( A ) camera of Hallmark gene set responses associated with bone metastasis ( bar is - log10FDR ; false discovery rate ) . Color indicates mean log2fold change ( FC ) of genes in gene set . Bar width is relative gene set . Dashed shows FDR = 0 . 05 ; ( B ) barcode plot showing enrichment of Hallmark IFN - α response gene set in genes suppressed in bone metastases , using signed log - likelihood statistic ( EdgeR ) . Bars indicate value of statistic for each gene in gene set ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Bulk RNA-seq analysis of DE genes (DEG) significantly suppressed (FDR < 0.05; generalized linear model extraction (GLM) by edgeR) in proliferating RM1 bone-derived (BD) cells compared to parental RM1 and lung metastases. Tumor-intrinsic type I IFN signaling is suppressed in proliferating bone lesions (n = 1) compared to parental RM1 (n = 2) cells as shown by (A) camera analysis of Hallmark gene set responses associated with bone metastasis (bar length is the -log10FDR; false discovery rate). Color indicates mean log2fold change (FC) of all genes in gene set. Bar width is relative gene set size. Dashed line shows FDR = 0.05; (B) barcode plot showing enrichment of the Hallmark IFN-α response gene set in genes suppressed in bone metastases, using the signed log-likelihood ratio statistic (EdgeR). Bars indicate value of the statistic for each gene in the gene set;


( C ) log2FC values of key downregulated IRGs , with genes retained in dormant classical downstream IFN signaling targets indicated . Altered IRGs directly involved in IFN - α / β production ( blue ) compared to downstream IFN targets ( blue ) are segregated . p values represented as * < 0 . 05 , * * < 0 . 005 * * * < 0 . 0005 ; GLM by edgeR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) log2FC values of key downregulated IRGs, with genes retained in dormant cells and classical downstream IFN signaling targets indicated. Altered IRGs directly involved in IFN-α/β production (light blue) compared to downstream IFN targets (dark blue) are segregated. p values represented as * < 0.05, ** < 0.005 and *** < 0.0005; GLM by edgeR.


( E ) of mean normalized voom expression of IRGs suppressed in RM1 bone metastases ( n = 1 ) compared to lung metastases ( n = 1 ) parental ( n = 2 ) .

O O O O O O O O O O O O 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) Heatmap of mean normalized voom expression of IRGs suppressed in RM1 bone metastases (n = 1) compared to lung metastases (n = 1) and parental cells (n = 2).


( F ) qRT - PCR validation of Irf7 Irf9 downregulation in RM1 from bone metastases ( RMI BD ) compared to parental RM1 , lung metastases ( RM1 lung ) naïve bone marrow ( BM ) ( n = 3 mice per group ) . p values represented as * * < 0 . 005 * * * < 0 . 0005 ( Student ' s t ) .

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

(F) qRT-PCR validation of Irf7 and Irf9 downregulation in RM1 cells from bone metastases (RMI BD) compared to parental RM1 cells, lung metastases (RM1 lung) and naïve bone marrow (BM) (n = 3 mice per group). p values represented as ** < 0.005 and *** < 0.0005 (Student's t test).


( G ) qRT - PCR of Irf7 Irf9 downregulation in parental RM1 , RM1 from bone metastases ( RM1 BD ) in WT Ifnar1 deficient ( - / - ) mice , with naïve BM from WT Ifnar1 ( - / - ) for reference . ( n = 3 mice per group ; n = 1 for - / - BM control ) . p values represented as * < 0 . 05 * * < 0 . 005 ( Student ' s t ) .

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

(G) qRT-PCR of Irf7 and Irf9 downregulation in parental RM1 cells, RM1 cells from bone metastases (RM1 BD) in WT and Ifnar1 deficient (-/-) mice, with naïve BM from WT and Ifnar1 (-/-) animals for reference. (n = 3 mice per group; n = 1 for -/- BM control). p values represented as * < 0.05 and ** < 0.005 (Student's t test).


( A ) Stability of Irf7 Irf9 mRNA suppression by qRT - PCR in ex vivo bone - derived ( RM1 BD Irf - ; n = 7 ) in culture compared to a bone - derived that showed initial loss during early passage ( RM1 BD REV ( EP ) ; n = 3 ) then reverted to parental expression at late passage ( RM1 BD REV ( LP ) ; n = 4 ) compared to parental RM1 ( n = 3 ) .

O O O O O B-MEASURED_VAR I-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

(A) Stability of Irf7 and Irf9 mRNA suppression by qRT-PCR in ex vivo bone-derived cells (RM1 BD Irf-; n = 7) in culture compared to a bone-derived line that showed initial loss during early passage (RM1 BD REV (EP); n = 3) then reverted to parental expression levels at late passage (RM1 BD REV (LP); n = 4) compared to parental RM1 (n = 3).


( B ) ELISA of IFN - α production by RM1 parental ( n = 3 ) , RM1 bone - derived Irf low ( RM1 BD Irf - ; n = 4 ) RM1 BD REV ( n = 3 ) subsequent to poly I : C stimulation .

O O 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 O O O 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) ELISA of IFN-α production by RM1 parental (n = 3), RM1 bone-derived Irf low (RM1 BD Irf-; n = 4) and RM1 BD REV cells (n = 3) subsequent to poly I:C stimulation.


( C ) qRT - PCR of Irf7 Irf9 expression in RM1 BD Irf - ± 48 h treatment with MS275 ( 1 μM ) ( n = 7 - 9 ) .

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

(C) qRT-PCR analysis of Irf7 and Irf9 expression in RM1 BD Irf- cells ± 48 h treatment with MS275 (1 μM) (n = 7-9).


( e ) qRT - PCR of Irf7 Irf9 expression in parental RM1 ( n = 4 ) 48 h post - contact culture with naïve BM ( n = 6 ) .

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

(e) qRT-PCR analysis of Irf7 and Irf9 expression in parental RM1 cells (n = 4) 48 h post-contact culture with naïve BM (n = 6).


( F ) qRT - PCR of Irf9 expression in parental RM1 ± 48 h co - culture with naïve BM under contact ( non - transwell ; NT ) transwell ( 0 . 4 μm filters that prevent contact ) conditions ( n = 6 - 8 per condition ) .

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

(F) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h co-culture with naïve BM under contact (non-transwell; NT) and transwell (0.4 μm filters that prevent cell contact) conditions (n = 6-8 per condition).


( G ) qRT - PCR of Irf9 expression in parental RM1 ± 48 h contact co - culture with naïve BM ± MS275 ( 1 μM ) ( n = 3 - 6 per condition ) .

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

(G) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h contact co-culture with naïve BM ± MS275 (1 μM) (n = 3-6 per condition).


( A ) Bone metastasis - free survival in WT C57BL / 6 mice harboring RM1 parental , RM1 BD Irf - RM1 BD REV ( LP ) tumors ( n = 5 per group ; * * p = 0 . 0043 by log - rank ( Mantel - Cox ) ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 parental, RM1 BD Irf- and RM1 BD REV (LP) tumors (n = 5 per group; **p = 0.0043 by log-rank (Mantel-Cox) test).


( C ) ELISA of IFN - α production by RM1 parental , RM1 BD Irf - base vector ( BV ) RM1 BD Irf7 over - expressing ( OE ) with 24 h poly I : C stimulation ( n = 4 ) .

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

(C) ELISA of IFN-α production by RM1 parental, RM1 BD Irf- base vector (BV) and RM1 BD Irf7 over-expressing (OE) with 24 h poly I:C stimulation (n = 4).


( D ) Bone metastasis - free survival in WT C57BL / 6 mice subsequent to IC inoculation of RM1 BD Irf - BV ( n = 9 ) RM1 BD Irf7 OE ( n = 7 ) ( * * p = 0 . 0028 by log - rank ( Mantel - Cox ) ) .

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

(D) Bone metastasis-free survival in WT C57BL/6 mice subsequent to IC inoculation of RM1 BD Irf- BV (n = 9) and RM1 BD Irf7 OE (n = 7) cells (**p = 0.0028 by log-rank (Mantel-Cox) test).


( E ) Bone metastasis - free survival in Ifnar1 - / - mice harboring RM1 BD Irf - BV RM1 BD Irf7 OE tumors ( n = 4 per group ) representative bioluminescent imaging of burden in leg bones ( also shown in Appendix Fig S4F ) .

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

(E) Bone metastasis-free survival in Ifnar1 -/- mice harboring RM1 BD Irf- BV and RM1 BD Irf7 OE tumors (n = 4 per group) and representative bioluminescent imaging of tumor burden in leg bones (also shown in Appendix Fig S4F).


( F ) Immunohistochemical ( ) for cerulean ( anti - eCFP ; green ) on naïve WT bone , RM1 parental , RM1 BD Irf - BV RM1 BD Irf7 OE - bearing bones derived from WT ; RM1 BD Irf7 OE - bearing bones derived from Ifnar1 - / - at survival endpoints . Blue represents DAPI . Scale bar , 100 μm .

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

(F) Immunohistochemical (IHC) staining for cerulean (anti-eCFP; green) on naïve WT bone, RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE tumor-bearing bones derived from WT animals; and RM1 BD Irf7 OE tumor-bearing bones derived from Ifnar1 -/- animals at survival assay endpoints. Blue represents DAPI nuclear staining. Scale bar, 100 μm.


( G - I ) FACS of dormant ( claret + ) RM1 BD Irf - RM1 BD Irf7 OE from bone at ( G ) day 11 post - IC injection ( n = 4 per group ) ( h ) day 17 ( n = 4 - 6 mice group / time point ) with ( I ) D17 active ( claret - ) colony quantitation ( mean + 1 ) determined by multiphoton imaging IMARIS interrogation ( n = 9 bones from 3 mice per condition ) . Median shown . Upper low box hinges denote first third quartiles . Whiskers mark value limits .

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

(G-I) FACS analysis of dormant (claret+) RM1 BD Irf- and RM1 BD Irf7 OE cells from bone at (G) day 11 post-IC injection (n = 4 per group) and (h) day 17 (n = 4-6 mice group/time point) with (I) D17 active (claret-) colony quantitation (mean +1) determined by multiphoton imaging and IMARIS interrogation (n = 9 bones from 3 mice per condition). Median shown. Upper and low box hinges denote first and third quartiles. Whiskers mark value limits.


( J ) FACS of IFN - γ + TNF - α + CD8 + T ( % ) post - ICS induction of T ( spleen - derived from - bearing mice ) activation by RM1 parental , RM1 BD Irf - BV RM1 BD Irf7 OE ( n = 6 per condition / group ) .

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

(J) FACS analysis of IFN-γ+ and TNF-α+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation by RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE cells (n = 6 per condition/group).


( K ) Mean quantitation of TRAP - stained osteoclasts differentiated with M - CSF ± RANKL in co - culture with RM1 BD Irf - BV RM1 BD Irf7 OE with representative wells shown ( n = 3 per condition M - CSF ; n = 6 per condition M - CSF + RANKL ; also shown in Appendix Fig S4J ) . Scale bar represents 100 μm .

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

(K) Mean quantitation of TRAP-stained osteoclasts differentiated with M-CSF ± RANKL in co-culture with RM1 BD Irf- BV and RM1 BD Irf7 OE cells with representative wells shown (n = 3 per condition M-CSF; n = 6 per condition M-CSF + RANKL; also shown in Appendix Fig S4J). Scale bar represents 100 μm.


( A ) ELISA of IFN - α production by RM1 BD Irf - ± single agent MS275 poly I : C treatment or 48 h pre - treatment with MS275 ( pMS ) prior to poly I : C stimulation ( n = 6 ) .

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

(A) ELISA of IFN-α production by RM1 BD Irf- cells ± single agent MS275 and poly I:C treatment or 48 h pre-treatment with MS275 (pMS) prior to poly I:C stimulation (n = 6).


( B ) Mean of H2 - Kb on RM1 BD Irf - by FACS ± MS275 , poly I : C or combination treatment ( n = 3 ) .

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

(B) Mean fluorescence intensity of H2-Kb staining on RM1 BD Irf- cells by FACS ± MS275, poly I:C or combination treatment (n = 3).


( C ) FACS of IFN - γ + CD8 + T ( % ) post - ICS induction of T ( spleen - derived from RM1 BD Irf - - bearing mice ) activation upon re - stimulation with RM1 MS275 , poly I : C or combination treatment , with NAC ( no antigen - presenting ) RM1 BD Irf7 OE controls ( n = 3 ; Irf7 OE control ( C ) , n = 1 )

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

(C) FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1)


FACS of IFN - γ + CD8 + T ( % ) post - ICS induction of T ( spleen - derived from RM1 BD Irf - - bearing mice ) activation upon re - stimulation with RM1 MS275 , poly I : C or combination treatment , with NAC ( no antigen - presenting ) RM1 BD Irf7 OE controls ( n = 3 ; Irf7 OE control ( C ) , n = 1 ) with ( D ) representative FACS plots shown .

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

FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1) with (D) representative FACS plots shown.


( E ) Bone metastasis - free survival in WT C57BL / 6 mice harboring RM1 BD Irf - ± MS275 , poly I : C or combination treatment ( n = 6 per group ; log - rank ( Mantel - Cox ) ) .

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

(E) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 BD Irf- cells ± MS275, poly I:C or combination treatment (n = 6 per group; log-rank (Mantel-Cox) test).


( F ) Mean total burden in bone ( femurs , tibias , spine humerus ) at endpoint by bioluminescent score ( log2 p / sec / cm2 / sr ) with values < 4 . 0x104 representing zero burden ( n = 24 bones per group ; 6 mice per group ) representative bioluminescent imaging of burden in spine ( shown in Appendix Figure S5E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mean total tumor burden in bone (femurs, tibias, spine and humerus) at endpoint by bioluminescent intensity score (log2 p/sec/cm2/sr) with values < 4.0x104 representing zero burden (n = 24 bones per group; 6 mice per group) and representative bioluminescent imaging of tumor burden in spine (all shown in Appendix Figure S5E).


( A , FACS of peripheral blood ( PB ) T NK lymphocyte representation activation

(A, FACS analysis of peripheral blood (PB) T and NK lymphocyte representation and activation status


B ) FACS of FoxP3 + CD4 + CD4 + effector memory T status at days 4 10 post - IC inoculation across treatment settings ( n = 4 - 6 per group ) .

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

B) FACS analysis of FoxP3+ CD4+ and CD4+ effector memory T cell status at days 4 and 10 post-IC tumor cell inoculation across treatment settings (n = 4-6 per group).


( C , Bioluminescent imaging ( C ) of a combination group mouse with clearance from day 7 to 41 ( full experimental cohort shown in Appendix Fig S5E )

O O O O O O 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, Bioluminescent imaging (C) of a combination group mouse with tumor clearance from day 7 to 41 (full experimental cohort shown in Appendix Fig S5E)


D ) post - IC inoculation with associated specific CD8 + memory T response . This is represented by FACS of IFN - γ + CD8 + T ( % ) post - ICS induction of T ( spleen - derived from - bearing mice ) activation upon re - stimulation with RM1 BD Irf - MS275 , poly I : C or combination treatments in vitro , with NAC RM1 BD Irf7 OE controls ( n = 3 ; Irf7 OE control ( C ) , n = 1 ) . Representative FACS plots shown on right .

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

D) post-IC inoculation with associated specific CD8+ memory T cell response. This is represented by FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation upon re-stimulation with RM1 BD Irf- cells and MS275, poly I:C or combination treatments in vitro, with NAC and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1). Representative FACS plots shown on right.


Compositional of - seq data ( FDR < 0 . 05 ; GLM by edgeR ) from bone metastases ( n = 9 ) tumors ( n = 12 ) revealed ( A ) immune ( B ) were relative ( % of total ) across samples , with a mean purity > 85 % as determined by ARMET . Median shown . Upper low box hinges denote first third quartiles . Upper lower whiskers mark values ± 1 . 5IQR from hinge . Data points beyond whiskers are shown individually . p values not significant by Wilcoxon rank sum ( R ) .

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

Compositional analysis of RNA-seq data (FDR < 0.05; GLM by edgeR) from bone metastases (n = 9) and primary tumors (n = 12) revealed (A) immune cells and (B) tumor cells were relative (% of total) across all samples, with a mean tumor purity >85% as determined by ARMET. Median shown. Upper and low box hinges denote first and third quartiles. Upper and lower whiskers mark the values ± 1.5IQR from the hinge. Data points beyond the whiskers are shown individually. p values not significant by Wilcoxon rank sum test (R).


Hallmark gene set responses associated with bone metastasis by camera shows ( C ) enrichment of type I ( IFN - α ) IFN responses in suppressed genes ( barcode plot ) .

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

Hallmark gene set responses associated with bone metastasis by camera analysis shows (C) enrichment of type I (IFN-α) IFN responses in suppressed genes (barcode plot).


( D ) of type I IFN DE IRGs ( normalized log2CPM ; per million ) significantly suppressed in bone metastases ( n = 9 ) compared to ( n = 12 ) benign ( n = 3 ) tumors , grouped by type I IFN specific , type II III IFN co - regulation status . Scale truncated at ± 5 relative log2CPM . GLM by edgeR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap of type I IFN DE IRGs (normalized log2CPM; counts per million) significantly suppressed in bone metastases (n = 9) compared to primary (n = 12) and benign (n = 3) tumors, grouped by type I IFN specific, and type II and III IFN co-regulation status. Scale truncated at ± 5 relative log2CPM. GLM by edgeR.


( E ) for IRF9 expression in prostate tumors bone metastases with matched samples indicated . IRF9 indicated by brown ( DAB ) . Scale bar represents 100 μm . Full - face slides shown in Appendix Fig S7C .

O O O O B-MEASURED_VAR 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

(E) IHC for IRF9 expression in primary prostate tumors and bone metastases with matched samples indicated. IRF9 indicated by brown (DAB) staining. Scale bar represents 100 μm. Full-face slides shown in Appendix Fig S7C.


( F ) of HLA genes ( normalized log2CPM ) suppressed ( p < 0 . 05 ; FDR < 0 . 05 ) in bone metastases ( n = 9 ) , tumors ( n = 12 ) benign tumors ( n = 3 ) . Scale truncated at ± 5 relative log2CPM . GLM by edgeR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap of HLA genes (normalized log2CPM) suppressed (p <0.05; FDR <0.05) in bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). Scale truncated at ±5 relative log2CPM. GLM by edgeR.


( G ) CIBERSORT of absolute leukocyte proportions ( arbitrary units , AU ) in mRNA samples of bone metastases ( n = 9 ) , tumors ( n = 12 ) benign tumors ( n = 3 ) . p values by Mann - Whitney U .

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(G) CIBERSORT analysis of absolute leukocyte proportions (arbitrary units, AU) in mRNA samples of bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). p values by Mann-Whitney U.


( H ) Kaplan Meier Curve of human prostate ( n = 499 from 498 TCGA samples ) biochemical recurrence on alterations in a 8 - IRG signature ( Appendix Fig S7D ) . groups stratified by mRNA z - Score ( - seq V2 RSEM ) ± 1 . 5 . Hazard 1 . 674 ( 95 % confidence interval ( CI ) 2 . 707 to 1 . 035 ) ; * p = 0 . 03 by log - rank .

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(H) Kaplan Meier Curve of human prostate (n = 499 from 498 TCGA samples) biochemical recurrence based on primary tumor alterations in a core 8-IRG signature (Appendix Fig S7D). Patient groups stratified by mRNA z-Score (RNA-seq V2 RSEM) ± 1.5. Hazard ratio 1.674 (95% confidence interval (CI) 2.707 to 1.035); *p = 0.03 by log-rank test.


Viability of single double mutants of RNAPII rad52Δ , mre11Δ cdc44 ‐ 8 . Tenfold serial dilutions of double mutants obtained by genetic crosses were tested for growth in plain SC medium or supplemented with 10 mM HU or 0 . 005 % MMS .

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Viability of single and double mutants of RNAPII and rad52Δ, mre11Δ and cdc44‐8. Tenfold serial dilutions of double mutants obtained by genetic crosses were tested for growth in plain SC medium or supplemented with 10 mM HU or 0.005% MMS.


Tetrad of rpb1 ‐ S751F , rad52Δ mre11Δ crosses . Δ indicates double mutants that fail to grow .

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Tetrad analysis of rpb1‐S751F, rad52Δ and mre11Δ crosses. Δ indicates double mutants that fail to grow.


Tetrad of rpb1 ‐ S751F crossed by pol32Δ rad51Δ . Δ indicates triple mutants that fail to grow .

Tetrad analysis of rpb1‐S751F crossed by pol32Δ and rad51Δ. Δ indicates triple mutants that fail to grow.


HU MMS sensitivity of different mutant combinations of rpb1 ‐ 1 , rpb1 ‐ S751F , pol32Δ rad51Δ .

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HU and MMS sensitivity of different mutant combinations of rpb1‐1, rpb1‐S751F, pol32Δ and rad51Δ.


Western blot against phosphorylated form of histone H2A in presence of 100 mM HU in asynchronously growing WT RNAPII mutant . Ponceau is shown as a loading control .

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Western blot against the phosphorylated form of histone H2A in the presence of 100 mM HU in asynchronously growing WT and RNAPII mutant cells. Ponceau staining is shown as a loading control.


Rad52 foci formation at 30 and 37°C.


Recombination frequencies between inverted repeats in WT RNAPII mutants measured with plasmid ‐ born pTINV system . Recombinants were selected as Leu + .

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Recombination frequencies between inverted repeats in WT and RNAPII mutants measured with the plasmid‐born pTINV system. Recombinants were selected as Leu+.


Recombination frequencies using direct ‐ repeat chromosomal system leu2 ‐ k : : ADE2 ‐ URA3 : : leu2 ‐ k in WT RNAPII mutants . Deletion events were detected quantified in media containing 5 ‐ fluorotic acid ( FOA ) could be directly visualized as red sectors .

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Recombination frequencies using the direct‐repeat chromosomal system leu2‐k::ADE2‐URA3::leu2‐k in WT and RNAPII mutants. Deletion events were detected and quantified in media containing 5‐fluorotic acid (FOA) and could be directly visualized as red sectors.


Recombination frequencies between plasmid ‐ born direct repeats in WT RNAPII mutants . frequency of Leu + recombination was determined in systems L ‐ lacZ GL ‐ lacZ . Recombination frequencies are plotted as a function of transcription . Low transcription refers to GL ‐ lacZ systems in strains cultured in 2 % glucose ; medium refers to L ‐ lacZ in 2 % glucose , high to GL ‐ lacZ in 2 % galactose .

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Recombination frequencies between plasmid‐born direct repeats in WT and RNAPII mutants. The frequency of Leu+ recombination was determined in the systems L‐lacZ and GL‐lacZ. Recombination frequencies are plotted as a function of the transcription levels. Low transcription refers to the GL‐lacZ systems in strains cultured in 2% glucose; medium refers to L‐lacZ in 2% glucose, and high to GL‐lacZ in 2% galactose.


Mitotic stability of centromeric plasmid pGAL ‐ lacZ in RNAPII mutants . Stability was analyzed in galactose after 23 generations . A small diagram of each system ( not drawn to scale ) is shown .

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Mitotic stability of centromeric plasmid pGAL‐lacZ in RNAPII mutants. Stability was analyzed in galactose after 23 generations. A small diagram of each system (not drawn to scale) is shown.


Recombination frequencies measured with pTINV system in WT , rpb1 ‐ 1 , mre11Δ rpb1 ‐ 1 mre11Δ .

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Recombination frequencies measured with the pTINV system in WT, rpb1‐1, mre11Δ and rpb1‐1 mre11Δ.


Rad52 focus formation in asynchronously growing at 30 ° C .

Rad52 focus formation in asynchronously growing cells at 30°C.


Viability after different times in 10 mM HU . same cultures without HU were used as a control . average values standard deviations from three independent experiments are shown .

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Viability after different times in 10 mM HU. The same cultures without HU were used as a control. The average values and standard deviations from three independent experiments are shown.


Rad52 focus formation in asynchronously growing expressing normal or high of RNH1 .

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Rad52 focus formation in asynchronously growing cells expressing normal or high levels of RNH1.


FACS of cycle progression in rpb mutants . were synchronized in G1 with α ‐ factor monitored at different times after release in presence of 40 mM HU .

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FACS analysis of cell cycle progression in rpb mutants. Cells were synchronized in G1 with α‐factor and monitored at different times after release in the presence of 40 mM HU.


Slowdown of RF progression through a chromosomal region . 2D gel electrophoresis of replication intermediates of 4 . 3 ‐ kb PstI fragments from WT RNAPII mutants in presence of 40 mM HU at different times after G1 release . A black arrow indicates accumulation of long Y molecules , a one indicates specific accumulation of X molecules .

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Slowdown of RF progression through a chromosomal region. 2D gel electrophoresis of replication intermediates of 4.3‐kb PstI DNA fragments from WT and RNAPII mutants in the presence of 40 mM HU at different times after G1 release. A black arrow indicates accumulation of long Y molecules, and a white one indicates the specific accumulation of X molecules.


of replication by BrdU incorporation . were synchronized in G1 with α ‐ factor released in presence of BrdU . Samples were taken at indicated times processed to measure BrdU incorporation by ChIP . Replication was analyzed in two regions , one covering early origin ARS508 SPF1 gene other late origin ARS603 YFL034W gene .

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Analysis of replication by BrdU incorporation. Cells were synchronized in G1 with α‐factor and released in the presence of BrdU. Samples were taken at the indicated times and processed to measure BrdU incorporation by ChIP analysis. Replication was analyzed in two DNA regions, one covering the early origin ARS508 and the SPF1 gene and the other the late origin ARS603 and the YFL034W gene.


combing of replication . Graphs indicate distribution of BrdU track ( kb ) at 60 120 min after α ‐ factor release in presence of 40 mM HU , replication speed ( kb / min ) determined with two time points analyzed inter ‐ origin ( kb ) at 60 min after release . median was applied .

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DNA combing analysis of replication. Graphs indicate the distribution of BrdU track length (kb) at 60 and 120 min after α‐factor release in the presence of 40 mM HU, the replication speed (kb/min) determined with the two time points analyzed and the inter‐origin distance (kb) at 60 min after release. The median test was applied.


Dephosphorylation of Rad53 after exposure to HU . WT , rpb1 ‐ 1 rpb1 ‐ S751F were synchronized in G1 with α ‐ factor . After G1 release , were exposed to 100 mM HU for 1 h , washed to remove HU cultured in HU ‐ free medium for indicated periods . Western blot against Rad53 , signal ( Rad53 ‐ P / total Rad53 ) FACS profiles are shown .

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Dephosphorylation of Rad53 after exposure to HU. WT, rpb1‐1 and rpb1‐S751F cells were synchronized in G1 with α‐factor. After G1 release, cells were exposed to 100 mM HU for 1 h, washed to remove HU and cultured in HU‐free medium for the indicated periods. Western blot against Rad53, signal quantification (ratio Rad53‐P/total Rad53 protein) and FACS profiles are shown.


repair of MMS damage . Chromosome VII species revealed by hybridization with ADE5 , 7 probes in pulsed ‐ field gel electrophoresis ( PFGE ) . Asynchronously growing WT rpb1 were exposed to 0 . 1 % MMS for 30 min , washed to remove MMS cultured in MMS ‐ free medium for indicated periods to allow repair . Repair was calculated as of FLC appearing after MMS treatment . FLC signal was quantified with respect to total signal of each lane . Data show mean SD of 3 independent experiments . NLC , nonlinear chromosome . FLC , full ‐ linear chromosome . CF , chromosome fragment resulting from 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 actively transcribed PMA1 gene in RNAPII mutants . ChIP of RNAPII in asynchronously growing WT , rpb1 ‐ 1 , rpb1 ‐ S751F , rpb2 ‐ 10 rpb9Δ at PMA1 gene . scheme of gene PCR ‐ amplified fragments are shown . Recruitment values were calculated from amount of present in each region , normalizing IP / input ratios , relative to amount of of intergenic region . Error bars indicate 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 of expression of PMA1 gene . was isolated from mid ‐ log phase cultures grown in YPD ‐ rich medium . As 32P ‐ labeled probes , an internal 776 ‐ bp PMA1 fragment an internal 513 ‐ bp SCR1 fragment obtained by PCR were used . Bars indicate 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.


of Rrm3 recruitment in WT rpb1 ‐ 1 . Rrm3 IP histogram bars in y ‐ axis show average signal log2 of loci enriched in immunoprecipitated fraction along indicated regions in x ‐ axis . When fulfills 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 average ORF plotted as Rrm3 of significant ChIP hits per segment . Each gene was divided into 10 equivalent segments from start end coordinates ( segments 2 - 10 ) . Two additional segments of same were considered upstream ( 5 ′ ) downstream ( 3 ′ ) of each ORF ( segments 1 12 , respectively ) . average signal log2 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 average ARS plotted as Rrm3 of significant ChIP hits per segment . Each ARS was divided into 11 equivalent segments from start end coordinates . average signal log2 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 of a pool of transfected after treatment with Cas9 - D10A ' nickase ' indicated combination of gRNAs ~ 200 bp product was centred around start of exon 1 of SLD5 gene . Efficient cutting of both template strands by Cas9 - nickase was indicated by production of a smeary PCR product ( gRNAs 2 + 3 ) , reflecting repair that mostly produced small deletions around nicking site combination of gRNAs 1 + 3 did not produce a smeary PCR , due to a polymorphism within PAM motif of 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 indicated were used to generate 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 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 were synchronised as indicated content was monitored throughout 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 indicated times used to isolate TAP - SLD5 by immunoprecipitation on IgG beads . indicated were monitored by immunoblotting . For reasons of space , samples A , S , M 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 ) were treated with 5 µM CB - 5083 as indicated ( p97i = p97 inhibitor ) . Extracts were then incubated with IgG beads to isolate GINS via TAP - SLD5 indicated factors were monitored by immunoblotting . Accumulation of CMG helicase upon inhibition of p97 was reflected by increased association of GINS with CDC45 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 in fixed mouse ES that were stained with Hoechst to reveal condensed patches of heterochromatic ( examples marked by arrows ; colocalization of HP1 condensed in 83 % of , 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 of mouse ES expressing GFP - SLD5 mCherry - PCNA Arrows indicate co - localisation on heterochromatic patches , which was observed in 14 % asynchronous ( n = 238 ) . GFP - SLD5 appeared disappeared with similar kinetics to mCherry - PCNA ( 100 % , 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 were treated with p97i as indicated then fixed stained with Hoechst . Arrows indicate accumulation of GFP - SLD5 on constitutive heterochromatin ( 53 % , 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 replisome components CLASPIN ( i ) CTF4 ( ii ) in GFP - SLD5 treated with p97i . Arrows indicate co - localisation of GFP - SLD5 patches with CLASPIN ( 95 % , n = 107 ) or CTF4 ( 91 % , n = 45 ) . Scale bars in 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 ) extracts were generated after indicated treatments 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 were subjected to indicated treatments . level of LRR1 VHL was assessed by immunoblotting . asterisk denotes a non - specific band . ( C ) For experiment described in ( B ) , TAP - SLD5 was isolated from extracts 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 ) activity of CUL2LRR1 was inhibited in GFP - SLD5 , by treatment with a combination of LRR1 siRNA MLN4924 Fixed were analysed Arrows indicate accumulation of GFP - SLD5 on constitutive heterochromatin ( 49 % , n = 125 ) . ( E ) were treated as in ( D ) localisation of GFP - SLD5 was compared with CLASPIN by immunofluorescence . Arrows indicate co - localisation of GFP - SLD5 CLASPIN on constitutive heterochromatin ( in 90 % 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 of mouse ES expressing GFP - SLD5 mCherry - PCNA , following inhibition of CUL2LRR1 . Arrows illustrate 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 of mouse ES expressing GFP - SLD5 mCherry - Histone H2B ( from CAG promoter at ROSA26 locus after inhibition of CUL2LRR1 . Arrows indicate heterochromatic patches of GFP - SLD5 that disappear during entry into mitosis ( 98 % , n = 97 ) . ( C ) Equivalent data to ( B ) but for untreated ( 100 % of untreated lacked GFP - SLD5 on mitotic , 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 CMG helicase from extracts of mouse ES . 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 were processed Arrows in ( i ) indicate accumulation of GFP - SLD5 on heterochromatin during interphase , whereas arrows in ( ii ) denote GFP - SLD5 on 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 stable expression from CAG promoter of indicated forms of TRAIP at ROSA26 locus in TRAIP - / - mouse ES . Asterisks indicate non - specific bands . ( D ) of of mitotic with GFP - SLD5 on mitotic chromosomes , after inhibition of CUL2LRR1 in with indicated genotypes . were fixed before .

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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 of UbG76V - GFP normalized to mRFP in hypodermis from of indicated timepoint ( in hours ) after L4 stage . have been exposed to indicated knockdown RNAi strains or a that only contains empty RNAi vector . * P < 0 . 001 , Student t - corrected for multiple comparisons at each time point using Holm - Sidek method . N = 20 per genotype timepoint . Error bars indicate .

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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 col - 19 promoter of UbG76V - GFP ( green ) mRFP ( red ) in C . elegans hypodermis from a single integrated transgene at L4 + 48 hours . Wild - type are shown . Bar , 50 microns . UbG76V - GFP is barely detectable . E , F Co - expression via sur - 5 promoter of UbG76V - GFP ( green ) mCherry ( red ) in C . elegans from two separate integrated transgenes ( with focus on intestine ) at L4 + 48 hours . Wild - type are shown . Bar , 50 microns . UbG76V - GFP is barely detectable . G , H Co - expression of UbG76V - GFP ( green ) mRFP ( red ) in C . elegans hypodermis at L4 + 48 hours in dop - 1 ( vs100 ) mutants . Bar , 50 microns . Abundant , stable UbG76V - GFP is observed . I , J Co - expression of UbG76V - GFP ( green ) mCherry ( red ) in C . elegans from sur - 5 promoter at L4 + 48 hours in dop - 1 ( vs100 ) mutants . Bar , 50 microns . Abundant , stable UbG76V - GFP is observed , particularly in 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 of UbG76V - GFP normalized to mRFP in hypodermis from of indicated timepoint ( in hours ) after L4 stage of indicated genotype . * P < 0 . 001 , Student t - corrected for multiple comparisons at each time point using Holm - Sidek method . N = 20 per genotype timepoint . Error bars indicate .

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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 of UbG76V - GFP normalized to mRFP in hypodermis of at L4 + 48 hours of then indicated genotypes . * * * P < 0 . 001 , ANOVA with Dunnetts posthoc comparison to wild - type control . N = 20 per genotype timepoint . Error bars indicate .

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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 of UbG76V - GFP normalized to mCherry ( both expressed by sur - 5 promoter ) in intestine of at L4 + 48 hours of then indicated genotypes . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to wild - type control . N = 20 per genotype timepoint . Error bars indicate .

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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 L4 + 48 hour stage , probed with antibodies recognizing , ubiquitin , GFP , RPN - 11 , multiple 20S proteasome subunits , or actin as a loading control . express UbG76V - GFP in their hypodermis . position of molecular markers is shown on left of each blot . position of UbG76V - GFP , as well as UbG76V - GFP with indicated number of additional ubiquitin moieties , on molecular , is indicated on right of each blot . Twenty were loaded per lane for each indicated genotype ( WT indicates wild type ) .

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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 L4 + 48 hour stage , probed annotated as in ( A ) . express UbG76V - GFP from sur - 5 promoter . Thirty 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 of UbG76V - GFP normalized to mRFP from lysates of transgenic nematodes at L4 + 48 hours that express these reporters in hypodermis . were exposed to containing indicated RNAi clone or an empty RNAi vector as a control . * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to empty RNAi vector control . N = 3 trials . Error bars indicate .

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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 turnover of chymotrypsin substrate ) from same lysates exposed to same RNAi as in ( C ) . * P < 0 . 05 , ANOVA with Dunnetts posthoc comparison to empty RNAi vector control . N = 3 trials . Error bars indicate .

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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 of UbG76V - GFP normalized to mRFP from lysates of transgenic nematodes at L4 + 48 hours that express these reporters in hypodermis . were of 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 .

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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 turnover of chymotrypsin substrate ) from 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 of live of indicated genotype assayed after 6 - hour exposure to heat shock . 100 % of control of same genotype but not given heat shock survived . * P < 0 . 01 , Student t - . N = 3 trials . Error bars indicate .

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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 of of indicated genotype containing of Poly ( Q ) 44 : : YFP at indicated time ( in days ) after L4 stage . * P < 0 . 01 , Student t - . N = 3 trials , 50 - 80 per trial per day . Error bars indicate .

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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 indicated xenobiotic stress response gene ( or UbG76V - GFP or mRFP ) detected in wild type versus dop - 1 mutants . Values are on - seq / EdgeR , with relative for each gene normalized to value observed in wild - type control . * * * * P < 0 . 0001 , * * P < 0 . 01 , * P < 0 . 05 FDR from EdgeR using a Benjamini Hochberg correction .

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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 of UbG76V - GFP normalized to ( B ) mRFP in hypodermis from L4 + 48 hour exposed to feeding RNAi for either an empty vector control or indicated xenobiotic stress gene . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA , Tukey ' s multiple comparison compared to control . N = 20 per genotype timepoint . Error bars indicate .

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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 of UbG76V - GFP normalized to ( C ) mCherry in intestine from L4 + 48 hour exposed to feeding RNAi for either an empty vector control or indicated xenobiotic stress gene . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA , Tukey ' s multiple comparison compared to control . N = 20 per genotype timepoint . Error bars indicate .

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 of live raised on indicated feeding RNAi ( or empty vector control ) assayed after 6 - hour exposure to heat shock . 100 % of control of same genotype but not given heat shock survived . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , ANOVA , Tukey ' s multiple comparison compared to wild type . N = 3 trials . Error bars indicate .

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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 of UbG76V - GFP normalized to mRFP in hypodermis from L4 + 48 hour of indicated genotype . * * * * P < 0 . 0001 , ANOVA , Tukey ' s multiple comparison compared to wild type . N = 20 per genotype timepoint . Error bars indicate .

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 of live of indicated genotype assayed after 6 - hour exposure to heat shock . 100 % of control of same genotype but not given heat shock survived . * * P < 0 . 01 , * P < 0 . 05 , ANOVA , Tukey ' s multiple comparison compared to wild type . N = 3 trials . Error bars indicate .

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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 of of indicated genotype containing of Poly ( Q ) 44 : : YFP at indicated time ( in days ) after L4 stage . * P < 0 . 001 , Student t - corrected for multiple comparisons at each time point using Holm - Sidek method . N = 3 trials , 50 - 80 per trial per day . Error bars indicate .

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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 of UbG76V - GFP normalized to mRFP in hypodermis from L4 + 48 hour of indicated genotype . * * * * P < 0 . 0001 , ANOVA , Tukey ' s multiple comparison compared to wild type . N = 20 per genotype . Error bars indicate .

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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 of UbG76V - GFP normalized to mRFP in hypodermis from either L4 + 24 hour ( left half of graph ) or L4 + 48 hour ( right half of graph ) of indicated genotype . were either continually grown on regular OP50 food ( gray bars ) or switched to PA14 food at L4 stage ( orange bars ) . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * * P < 0 . 01 , ANOVA , Tukey ' s multiple comparison compared to wild type . N = 20 per genotype . Error bars indicate .

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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 ( of live assayed each hour after PA14 exposure ) for 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 ) , 42 hours for skn - 1 ( tm3411 ) ( N = 33 ) . Eight wild - type six dop - 1 mutants were censored ( crawled up side of plate dessicated ) . media contained 50 µg / ml of 5 - fluoro - 2 ′ - deoxyuridine ( FUdR to prevent growth of egg offspring during experiment .

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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 of UbG76V - GFP normalized to mRFP in hypodermis from either L4 + 48 hour ( left half of graph ) or L4 + 72 hour ( right half of graph ) of indicated genotype . were either continually grown on regular OP50 food ( gray bars ) or switched to NGM plates containing 1 mM paraquat at L4 stage ( orange bars ) . * * * * P < 0 . 0001 , * * * P < 0 . 001 , ANOVA , Tukey ' s multiple comparison compared to wild type . # P < 0 . 001 ANOVA , Bonferoni multicomparison as indicated by bracketed lines . N = 20 per genotype . Error bars indicate . No UbG76V - GFP was detected in wild type at L4 + 72 hours on either OP50 or switched to NGM plates containing 1mM paraquat .

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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 of median IC50 of entire panel to that of each generated from a screen of 23 pediatric cancer lines . Rhabdoid lines ( red ) cluster towards left of graph indicating these lines are more sensitive to mithramycin . These results confirm a previously published screen ( Osgood et al . , 2016 ) .

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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 of median IC50 of entire panel to that of each generated from a published screen of 445 agents in 62 sarcoma lines ( Teicher et al . , 2015 ) . rhabdoid , G401 ( red ) , appears on left side of graph indicating this is more sensitive to mithramycin .

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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 Ewing sarcoma lines . RT lines ( black ) are sensitive to mithramycin treatment with a similar IC50 value as TC32 ES ( grey ) . RT 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 .

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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 dependent increase in γH2AX following 8 - hour exposure to etoposide in BT12 G401 RT ( A ) . Red bar indicates measured IC50 ( Figure 1C ) . Despite induction of damage , 15 μM etoposide does not lead to apoptosis as indicated by live imaging in presence of caspase 3 , 7 ( CC3 , 7 ) reagent that fluoresces with caspase activation ( B ) . Scale bar ( lower left ) : 150μm .

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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 dependent increase in γH2AX following 8 - hour exposure to mithramycin ( MMA ) in BT12 G401 RT ( C ) . Red bar indicates measured IC50 ( Figure 1C ) . 100 nM Mithramycin induces apoptosis at 8 - hours as measured by CC3 , 7 without presence of damage ( D ) . Scale bar ( lower left ) : 150μm .

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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 treated with 20nM MMA show a different phenotype than with 100nM . Beyond 24h of exposure , there is evidence of mesenchymal differentiation appearance of maturing adipocytes . Scale bar ( lower left ) : 150μm .

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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 dependent increase in γH2AX following 8 - hour exposure to EC8042 in BT12 G401 RT . Red bar indicates 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 ) ( G ) is same although different fields are shown . Scale bar ( lower left ) : 150μm .

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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 of lipid deposits following 20nM MMA or 75nM EC8042 treatment for 24 48 - hours . Please note , solvent control for 20nM MMA 75nM EC8042 is 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 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 SMARCE1 SWI / SNF subunits from in a time - dependent manner in BT12 rhabdoid ( A ) but not U20S osteosarcoma ( B ) . Western blot showing whole lysate ( Total ) , soluble ( CS ) , soluble ( NS ) , bound ( Chr ) fractions collected after exposure to solvent ( S ) or 100nM mithramycin for 8 or 18h probed for SWI / SNF subunits ( BRD9 , SMARCC1 or SMARCE1 ) or H3 ( fraction control ) 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 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 proteasome inhibitor ( bortezomib ) rescues loss of expression following 8 or 18 - hour mithramycin treatment . BT12 were treated for 8 or 18 - hours with solvent ( S ) , mithramycin ( M , 100nM ) , bortezomib ( PI , 2 . 5μM ) , or combination of 100nM mithramycin 2 . 5μM bortezomib ( C ) .

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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 genome as measure by immunoprecipitation qPCR ( ChIP - qPCR ) at previously described SWI / SNF target genes , MYT1 ( 8 - hour , P = 0 . 0001 ; 18 - hour P = 0 . 0001 ) CCND1 ( 8 - hour , P = 0 . 0001 ; 18 - hour P = 0 . 0001 ) . ChIP quantitation is input ( ng of immunoprecipitated / input * 100 ) determined by absolute quantitation of sheared 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 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 : immunoprecipitation qPCR ( ChIP - qPCR ) of H3K27me3 at MYT1 ( 8 - hour , P = 0 . 0001 ; 18 - hour P = 0 . 0001 ) CCND1 ( 8 - hour , P = 0 . 02 ; 18 - hour P = 0 . 0001 ) . H3K27me3 occupancy is increased in a time - dependent manner . ChIP quantitation is input ( ng of immunoprecipitated / input * 100 ) determined by absolute quantitation of sheared 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 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 - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in BT12 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 - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in G401 relative to loading control ( H3 ) . Re - expression of SMARCB1 following doxycycline treatment inhibits 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 - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in G401 relative to loading control ( H3 ) . Re - expression of SMARCB1 following doxycycline treatment inhibits mithramycin - dependent effects on H3K27me3

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A , B : Mithramycin displaces BRD9 SMARCE1 SWI / SNF subunits from in a time - dependent manner in G401 ( A ) but only BRD9 with SMARCB1 re - expression in G401 ( B ) . Western blot showing whole lysate ( Total ) , soluble ( CS ) , soluble ( NS ) , bound ( Chr ) fractions collected after exposure to solvent ( S ) or 100nM mithramycin for 8 or 18h probed for SWI / SNF subunits ( BRD9 , SMARCB1 or SMARCE1 ) or H3 ( fraction control ) 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 show a threshold of exposure that leads to irreversible growth inhibition . were exposed to 100nM MMA for indicated times followed by replacement with drug - free medium . Beyond 8h ( red ) of mithramycin exposure , do not recover proliferative potential exhibit a phenotype consistent with . 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 induction of apoptosis as measured by 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 activity antagonizes mithramycin activity in BT12 rhabdoid . Data represents dose response curves of mithramycin in BT12 following a 48h exposure in presence of siRNA silencing of PRC2 subunit EZH2 or treatment with 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 ( wild type SWI / SNF ) to mithramycin . Data represents dose response curves of mithramycin in BT12 following a 48h exposure in presence of siRNA silencing of 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 - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in SMARCB1 - silenced U2OS 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 ) 10e - 7 q - value threshold . of induced repressed genes in ( C ) . Labels indicate names of genes that meet logFC 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 ) 10e - 7 q - value threshold . of induced repressed genes in ( F ) . Labels indicate names of genes that meet logFC 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 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.


loss of SP1 expression is associated with a decrease in SWI / SNF occupancy of SP1 promoter an increase in H3K27me3 . Data represents ChIP - qPCR following 8 - hours or 18 - hours of 100nM mithramycin exposure immunoprecipitation of SMARCC1 ( 8 - hour , P = 0 . 0006 ; 18 - hour P = 0 . 0001 ) . ChIP quantitation is input ( ng of immunoprecipitated / input * 100 ) determined by absolute quantitation of sheared 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 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.