( B ) Individual ( left ) and average ( right ) in vivo growth of subcutaneous wild - type or sgRnf31 B16 - F10 ova tumors left untreated or treated with adoptively transferred OT - I CD8 + T cells ( on days indicated by black arrows ) ; * p < 0 . 05 , * * p < 0 . 01 , Unpaired t - test for OT - I - treated wild - type vs OT - I - treated sgRnf31 with n = 5 / 6 mice per group . Pooled data are represented as mean ± 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 O O O O O O O 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) Individual (left) and average (right) in vivo growth of subcutaneous wild-type or sgRnf31 B16-F10 ova tumors left untreated or treated with adoptively transferred OT-I CD8+ T cells (on days indicated by black arrows); *p < 0.05, **p < 0.01, Unpaired t-test for OT-I-treated wild-type vs OT-I-treated sgRnf31 with n = 5/6 mice per group. Pooled data are represented as mean ± SEM.


( C ) Overall survival of mice from ( B ) ; * p < 0 . 05 , * * p < 0 . 01 , Log - rank test .

O O O O O O O O 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) Overall survival of mice from (B); *p < 0.05, **p < 0.01, Log-rank test.


( E and F ) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with ethanol - d6 ( 5 mM ) or ethanol - 1 - 13C ( 5 mM ) for 0 . 5 h . Relative acetate m + 3 and acetate m + 1 levels ( E - F , top panels ) were examined . Protein levels of p53 and ALDH2 ( E - F , bottom panels ) were analyzed by western blot .

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

(E and F) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with ethanol-d6 (5 mM) or ethanol-1-13C (5 mM) for 0.5 h. Relative acetate m+3 and acetate m+1 levels (E-F, top panels) were examined. Protein levels of p53 and ALDH2 (E-F, bottom panels) were analyzed by western blot.


( H - J ) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with doxorubicin ( 2 ng / mL ) ( H ) nutlin3α ( 10 μM ) ( I ) or cyanamide ( 1 mM ) ( J ) for 24 h . Relative enzymatic activity of ALDH2 was examined ( top panel ) . Protein expression was determined by western blot analysis ( bottom panel ) .

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 B-CONTROLLED_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

(H-J) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with doxorubicin (2 ng/mL) (H) nutlin3α (10 μM) (I) or cyanamide (1 mM) (J) for 24 h. Relative enzymatic activity of ALDH2 was examined (top panel). Protein expression was determined by western blot analysis (bottom panel).


( P - R ) p53 + / + and p53 - / - mice were injected intraperitoneally with ethanol - d6 ( 2 g / kg ) for 0 . 5 h . Relative acetate m + 3 levels in mouse liver tissues ( P ) and mouse serum ( Q ) , and relative hepatic acetyl - CoA ( R ) were examined .

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

(P-R) p53+/+ and p53-/- mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 0.5 h. Relative acetate m+3 levels in mouse liver tissues (P) and mouse serum (Q), and relative hepatic acetyl-CoA (R) were examined.


( S ) Mouse hepatocyte isolated from p53 + / + and p53 - / - mice and lysates were treated with or without cyanamide ( 1mM ) . Relative acetyl - CoA level ( top panel ) and protein expression ( bottom panel ) were measured . ( T ) CCC - HEL - 1 cells transfected with control siRNA or p53 siRNA were treated with or without cyanamide ( 1mM ) . Relative acetyl - CoA level ( top panel ) and protein expression ( bottom panel ) were analyzed .

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 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 B-CONTROLLED_VAR 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

(S) Mouse hepatocyte isolated from p53+/+ and p53-/- mice and lysates were treated with or without cyanamide (1mM). Relative acetyl-CoA level (top panel) and protein expression (bottom panel) were measured. (T) CCC-HEL-1 cells transfected with control siRNA or p53 siRNA were treated with or without cyanamide (1mM). Relative acetyl-CoA level (top panel) and protein expression (bottom panel) were analyzed.


( U - X ) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were transfected with control siRNA or ALDH2 siRNA and were treated with ethanol - d6 ( 5 mM ) ( U ) or ethanol - 1 - 13C ( 5 mM ) ( X ) for 0 . 5 h . ( U ) Relative acetate m + 3 and acetyl - CoA m + 3 levels are shown . ( X ) Relative acetate m + 1 and acetyl - CoA m + 3 levels were examined . Protein levels of p53 and ALDH2 were analyzed by western blot ( V - W ) .

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

(U-X) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were transfected with control siRNA or ALDH2 siRNA and were treated with ethanol-d6 (5 mM) (U) or ethanol-1-13C (5 mM) (X) for 0.5 h. (U) Relative acetate m+3 and acetyl-CoA m+3 levels are shown. (X) Relative acetate m+1 and acetyl-CoA m+3 levels were examined. Protein levels of p53 and ALDH2 were analyzed by western blot (V- W).


( C ) Lysates from mice liver tissues ( WT , p53 - / - and ALDH2 - / - ) were immunoprecipitated using p53 or ALDH2 antibody , and bound proteins were analyzed by western blot .

O O O O O O O O O O O O O O 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) Lysates from mice liver tissues (WT, p53-/- and ALDH2-/-) were immunoprecipitated using p53 or ALDH2 antibody, and bound proteins were analyzed by western blot.


( G ) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin - resistant ALDH2 mut312 - 323 in ALDH2 - knockout cell lines . The cells were then transfected with control siRNA or p53 siRNA for 48 h and treated with ethanol ( 5 mM ) for 0 . 5 h and relative ALDH2 enzymatic activities were examined . Protein expression was analyzed by western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2 mut312-323 in ALDH2-knockout cell lines. The cells were then transfected with control siRNA or p53 siRNA for 48 h and treated with ethanol (5 mM) for 0.5 h and relative ALDH2 enzymatic activities were examined. Protein expression was analyzed by western blot.


( H ) Lysates from 293T cells transfected with HA - ALDH2 , Flag - ALDH2 and increasing amounts of GST - p53 or vector control ( - ) as indicated were immunoprecipitated using anti - Flag M2 affinity gels , and bound proteins were analyzed by western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysates from 293T cells transfected with HA-ALDH2, Flag-ALDH2 and increasing amounts of GST-p53 or vector control (-) as indicated were immunoprecipitated using anti-Flag M2 affinity gels, and bound proteins were analyzed by western blot.


( I ) HepG2 cells infected with lentivirus expressing ALDH2 sgRNA or control sgRNA were treated with ethanol ( 5 mM ) for 0 . 5 h and cell lysates were treated with disuccinimidyl suberate ( DSS ) or DMSO for 0 . 5 h and protein levels of p53 and ALDH2 were examined .

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

(I) HepG2 cells infected with lentivirus expressing ALDH2 sgRNA or control sgRNA were treated with ethanol (5 mM) for 0.5 h and cell lysates were treated with disuccinimidyl suberate (DSS) or DMSO for 0.5 h and protein levels of p53 and ALDH2 were examined.


( J ) Subcellular fractionation of HepG2 cells treated with doxorubicin ( 2 ng / mL ) or nutlin3α ( 10 μM ) or PFTα ( 10 μM ) for 24 h were immunoprecipitated using p53 antibody , and bound proteins were analyzed by western blot .

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 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

(J) Subcellular fractionation of HepG2 cells treated with doxorubicin (2 ng/mL) or nutlin3α (10 μM) or PFTα (10 μM) for 24 h were immunoprecipitated using p53 antibody, and bound proteins were analyzed by western blot.


( A - B ) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA under glucose or pyruvate deprivation and cell lysates were used to measure ALDH2 activities ( A , B , top panels ) and examine p53 and ALDH2 proteins ( A , B , bottom panels ) .

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

(A-B) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA under glucose or pyruvate deprivation and cell lysates were used to measure ALDH2 activities (A, B, top panels) and examine p53 and ALDH2 proteins (A, B, bottom panels).


( E ) CETSAs exhibit the binding affinity of pyruvate to the N186 amino acid in HepG2 cells . Cell lysates were separated by SDS - PAGE and analyzed by western blot . Actin was used as an internal control . Relative ALDH2 band intensities were quantified and plotted against corresponding incubation temperatures .

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

(E) CETSAs exhibit the binding affinity of pyruvate to the N186 amino acid in HepG2 cells. Cell lysates were separated by SDS-PAGE and analyzed by western blot. Actin was used as an internal control. Relative ALDH2 band intensities were quantified and plotted against corresponding incubation temperatures.


( F ) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin - resistant ALDH2 - N186A , E285A , C319A in ALDH2 - knockout cell lines . Cells were digested and cell lysates were incubated with the indicated doses of pyruvate for 1 hour on ice and 0 . 5 hour at room temperature , followed by pronase digestion . ALDH2 proteins were analyzed by western blot . Relative ALDH2 / Actin ratios are shown .

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

(F) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2-N186A, E285A, C319A in ALDH2-knockout cell lines. Cells were digested and cell lysates were incubated with the indicated doses of pyruvate for 1 hour on ice and 0.5 hour at room temperature, followed by pronase digestion. ALDH2 proteins were analyzed by western blot. Relative ALDH2/Actin ratios are shown.


( H - J ) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin - resistant ALDH2 N186A in ALDH2 - knockout cell lines . The cells were treated with pyruvate ( 1 mM ) for 24 h . Relative ALDH2 activity ( H , top panel ) , acetate ( I ) and acetyl - CoA ( J ) were measured . The expression of p53 and ALDH2 were analyzed by western blot ( H , bottom panel ) .

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

(H-J) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2 N186A in ALDH2-knockout cell lines. The cells were treated with pyruvate (1 mM) for 24 h. Relative ALDH2 activity (H, top panel), acetate (I) and acetyl-CoA (J) were measured. The expression of p53 and ALDH2 were analyzed by western blot (H, bottom panel).


H & E ( hematoxylin - eosin ) staining ( C , top ) and oil red O staining ( C , bottom ) were examined .

O O O 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&E (hematoxylin-eosin) staining (C, top) and oil red O staining (C, bottom) were examined.


Relative mRNA levels of IL - 6 , were analyzed . were examined . Data in are from n = 5 mice per group .

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

Relative mRNA levels of IL-6, were analyzed. were examined. Data in are from n=5 mice per group.


Relative mRNA levels of , IL - 1β , TNF - α , Cxcl1 , Cxcl2 , Col1a1 were analyzed . Data in are from n = 5 mice per group .

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

Relative mRNA levels of , IL-1β, TNF-α, Cxcl1, Cxcl2, Col1a1 were analyzed. Data in are from n=5 mice per group.


( J ) Relative triglyceride level was examined . ( K - L ) Relative levels of serum ALT ( K ) and AST ( L ) were examined . Data in are from n = 5 mice per group .

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

(J) Relative triglyceride level was examined. (K-L) Relative levels of serum ALT (K) and AST (L) were examined. Data in are from n=5 mice per group.


( M - O ) WT , p53 − / − , ALDH2 − / − and p53 − / − ALDH2 − / − C57BL / 6N male mice were injected intraperitoneally with ethanol - d6 ( 2 g / kg ) for 0 . 5 h as shown in Figure 1N . Relative acetate and acetate m + 3 levels in mouse liver tissues ( M ) and mouse serum ( N ) were examined . ( O ) Relative acetyl - CoA and acetyl - CoA m + 3 levels in mouse liver tissues were measured .

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

(M-O) WT, p53−/−, ALDH2−/− and p53−/−ALDH2−/− C57BL/6N male mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 0.5 h as shown in Figure 1N. Relative acetate and acetate m+3 levels in mouse liver tissues (M) and mouse serum (N) were examined. (O) Relative acetyl-CoA and acetyl-CoA m+3 levels in mouse liver tissues were measured.


( C - D ) p53 + / + and p53 - / - mice were injected intraperitoneally with ethanol - d6 ( 2 g / kg ) for 1 h . Chromatin fractions of liver tissues were subjected to ChIP - seq assay using H3K9ac antibody ( left panel ) , H3K27ac antibody ( right panel ) . Heat map of H3K9ac / H3K27ac binding signal ( rows ) from - 1 kb to + 1 kb surrounding the centre of H3K9ac / H3K27ac - binding sites for ChIP - seq tags ( C ) . Heatmap of H3K9ac / H3K27ac binding signal ( rows ) from - 1 kb to + 1 kb surrounding the centre of H3K9ac / H3K27ac - binding sites for ChIP - seq tags of fatty acid metabolism related genes ( D ) . For ChIP - seq analysis , there were no technical replicates for anti - H3K9ac or anti - H3K27ac .

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

(C-D) p53+/+ and p53-/- mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 1 h. Chromatin fractions of liver tissues were subjected to ChIP-seq assay using H3K9ac antibody (left panel), H3K27ac antibody (right panel). Heat map of H3K9ac/H3K27ac binding signal (rows) from -1 kb to +1 kb surrounding the centre of H3K9ac/H3K27ac-binding sites for ChIP-seq tags (C). Heatmap of H3K9ac/H3K27ac binding signal (rows) from -1 kb to +1 kb surrounding the centre of H3K9ac/H3K27ac-binding sites for ChIP-seq tags of fatty acid metabolism related genes (D). For ChIP-seq analysis , there were no technical replicates for anti-H3K9ac or anti-H3K27ac.


( F ) ChIP assays with H3K9ac antibody and H3K27ac antibody or IgG in WT and p53 − / − mice injected intraperitoneally with ethanol - d6 ( 2 g / kg ) or saline for 1 h . Bound SCD1 response elements ( RE ) were analyzed by qRT - PCR . Data in F are from n = 3 biological independent samples .

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 O O 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) ChIP assays with H3K9ac antibody and H3K27ac antibody or IgG in WT and p53−/− mice injected intraperitoneally with ethanol-d6 (2 g/kg) or saline for 1 h. Bound SCD1 response elements (RE) were analyzed by qRT-PCR. Data in F are from n=3 biological independent samples.


WT , p53 − / − , ALDH2 − / − and p53 − / − ALDH2 − / − C57BL / 6N male mice were fed control diet and ethanol diet as shown in Fig . 4A . Relative mRNA level of SCD1 ( G , top panel ) was analyzed . Protein levels of p53 , ALDH2 , SCD1 , Histone H3 and Ac - H3 , caspase 3 , cleaved caspase 3 , p21 ( G , bottom panel ) in mice liver tissues were examined . Data in G are from n = 5 technical replicates from one of three independent experiments with similar results .

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

WT, p53−/−, ALDH2−/− and p53−/−ALDH2−/− C57BL/6N male mice were fed control diet and ethanol diet as shown in Fig.4A. Relative mRNA level of SCD1 (G, top panel) was analyzed. Protein levels of p53, ALDH2, SCD1, Histone H3 and Ac-H3, caspase 3, cleaved caspase 3, p21 (G, bottom panel) in mice liver tissues were examined. Data in G are from n=5 technical replicates from one of three independent experiments with similar results.


WT and p53 − / − mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein . After 7 days , WT and p53 − / − mice were treated as described in the methods to establish a mouse model of chronic - binge ethanol consumption . ( C ) Protein levels of p53 and SCD1 were examined by western blot . level were examined .

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

WT and p53−/− mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein. After 7 days, WT and p53−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. (C) Protein levels of p53 and SCD1 were examined by western blot. level were examined.


WT and p53 − / − mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein . After 7 days , WT and p53 − / − mice were treated as described in the methods to establish a mouse model of chronic - binge ethanol consumption . Relative hepatic triglyceride level ( D ) , relative serum ALT ( E , left panel ) and AST ( E , right panel ) level were examined .

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

WT and p53−/− mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein. After 7 days, WT and p53−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. Relative hepatic triglyceride level(D), relative serum ALT (E, left panel) and AST (E, right panel) level were examined.


WT , ALDH2 − / − and p53 - / - ALDH2 − / − mice were injected with adeno - associated virus expressing AAV - GFP or AAV - SCD1 via tail vein . After 7 days , WT , ALDH2 − / − and p53 - / - ALDH2 − / − mice were treated as described in the methods to establish a mouse model of chronic - binge ethanol consumption . ( H ) Protein levels of p53 and SCD1 are shown .

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

WT, ALDH2−/− and p53-/-ALDH2−/− mice were injected with adeno-associated virus expressing AAV-GFP or AAV-SCD1 via tail vein. After 7 days, WT, ALDH2−/− and p53-/-ALDH2−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. (H) Protein levels of p53 and SCD1 are shown.


WT , ALDH2 − / − and p53 - / - ALDH2 − / − mice were injected with adeno - associated virus expressing AAV - GFP or AAV - SCD1 via tail vein . After 7 days , WT , ALDH2 − / − and p53 - / - ALDH2 − / − mice were treated as described in the methods to establish a mouse model of chronic - binge ethanol consumption . Relative hepatic triglyceride level ( I ) , relative serum ALT ( J , left panel ) and AST ( J , right panel ) were examined .

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

WT, ALDH2−/− and p53-/-ALDH2−/− mice were injected with adeno-associated virus expressing AAV-GFP or AAV-SCD1 via tail vein. After 7 days, WT, ALDH2−/− and p53-/-ALDH2−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. Relative hepatic triglyceride level (I), relative serum ALT (J, left panel) and AST (J, right panel) were examined.


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 B-MEASURED_VAR 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 .

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

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 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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 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

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 O O O O O O O B-CONTROLLED_VAR 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 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 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

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 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 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

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 .

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

H: Mesenchymal differentiation confirmed with oil red O staining of lipid deposits following 20nM MMA or 75nM EC8042 treatment for 24 and 48-hours. Please note, solvent control for 20nM MMA and 75nM EC8042 is the same. Scale bar (lower left): 150μm.


I : PPARγ mRNA expression is induced following 20nM mithramycin treatment as measured by qPCR fold - change relative to GAPDH ( 2ddCT ) . * * , P = 0 . 002 , * * * * , P = 0 . 0001 . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons .

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

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 ) .

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 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 O O O O O O O O O

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 .

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

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 ) .

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

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 .

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

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 .

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

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 ) .

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

Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in BT12 cells relative to loading control (H3).


Western blot showing concentration - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in G401 cells relative to loading control ( H3 ) . Re - expression of SMARCB1 following doxycycline treatment inhibits the mithramycin - dependent effects on H3K27me3 amplification

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

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

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


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 ) .

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

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 .

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 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

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 .

O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O

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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Suppression of SMARCB1 sensitizes U2OS osteosarcoma cells (wild type SWI/SNF) to mithramycin. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the SWI/SNF subunit SMARCB1 relative to a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments.


G . Western blot showing concentration - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in SMARCB1 - silenced U2OS cells relative to loading control ( H3 ) . Knockdown of SMARCB1 following siRNA suppression triggers mithramycin - dependent H3K27me3 amplification , while siNegative ( control ) does not have an effect on H3K27me3 following mithramycin exposure .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

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

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 .

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

The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of SMARCC1 (8-hour, P= 0.0006; 18-hour P=0.0001) . ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.


I : The loss of SP1 expression is associated with a decrease in SWI / SNF occupancy of the SP1 promoter and an increase in H3K27me3 . Data represents ChIP - qPCR analysis following 8 - hours or 18 - hours of 100nM mithramycin exposure and immunoprecipitation of H3K27me3 ( 8 - hour , P = 0 . 009 ; 18 - hour P = 0 . 0001 ) ChIP quantitation is percent input ( ng of immunoprecipitated DNA / input DNA * 100 ) determined by absolute quantitation of sheared chromatin relative to a standard curve . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons .

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

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 .

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

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 .

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

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 .

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

F: IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI/SNF loss (Erkek et al., 2019). CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8-hours and 18-hours mithramycin treatment.


G : IGV tracks of genes identified to have an increase in accessibility , H3K27ac and gene expression following mithramycin exposure . BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation , respectively .

O 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

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 .

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 B-MEASURED_VAR 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

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 ) .

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 O O O O O O O O O O O

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 ) .

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

C: Kaplan-Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3-day continuous infusions of EC8042 in (B). The shaded box indicates the duration of treatment. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).


D : 20X image of section of G401 treated tumors stained for H3K27me3 , cleaved caspase 3 , and γH2AX . The sections compare vehicle to treatment started on day 1 with 30 mg / kg of EC8042 administered continuously for 72 - hours ( 3 - day pump ) . H3K27me3 increases and correlates with apoptosis ( CC3 ) , however induction of CC3 is modest . γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects . Scale bar ( lower left ) : 50μm .

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

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 .

O O O O 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 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

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.


LL - 37 ( 4 kD ) detection by Western blotting . Prior to loading , samples were centrifuged through 10 kD ultrafiltration membranes and the equal amount of ultrafiltrate ( 19μg / lane ) subject to immunoblotting . High - molecular - weight proteins ( GAPDH ) were not detected in ultrafiltrate .

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

LL-37(4 kD) detection by Western blotting. Prior to loading, samples were centrifuged through 10 kD ultrafiltration membranes and the equal amount of ultrafiltrate (19μg/lane) subject to immunoblotting. High-molecular-weight proteins (GAPDH) were not detected in ultrafiltrate.


The CFU of PAO1 was measured by culturing in ultrafiltrate ( upper panel ) or retentate ( lower panel ) of mouse BALF samples from indicated mice . Initial additions of PAO1 were 1 × 103 CFU . Coculture duration , 6h . n = 3 .

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

The CFU of PAO1 was measured by culturing in ultrafiltrate (upper panel) or retentate (lower panel) of mouse BALF samples from indicated mice. Initial additions of PAO1 were 1 × 103 CFU. Coculture duration, 6h. n=3.


The bacterial CFU ( per gram ) in lungs of indicated mice with and without PAO1 infection ( 5 × 106 CFU ) for 6 , 24 and 48 hours . n = 3 . Error bars , S . E . M .

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

The bacterial CFU (per gram) in lungs of indicated mice with and without PAO1 infection (5 × 106 CFU) for 6, 24 and 48 hours. n=3. Error bars, S.E.M.


Representative histological sections of indicated lungs with PAO1 infection ( 5 × 106 CFU ) for 6 hours . H & E staining . Scale bar , 1000 μm ( upper panel ) and 50 μm ( lower panel ) .

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

Representative histological sections of indicated lungs with PAO1 infection (5 × 106 CFU) for 6 hours. H&E staining. Scale bar, 1000 μm (upper panel) and 50 μm (lower panel).


Histopathological injury score of indicated mouse lungs with PAO1 infection ( 5 × 106 CFU ) for 6 h and 24 h based on blinded expert judgement . n = 3 . Error bars , S . E . M .

Histopathological injury score of indicated mouse lungs with PAO1 infection (5 × 106 CFU) for 6 h and 24 h based on blinded expert judgement. n=3. Error bars, S.E.M.


Gene expression level of IL - 1β and IL - 6 of indicated mouse lung with PAO1 infection ( 5 × 106 CFU ) for 6 h and 24 h . n ≥ 3 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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

Gene expression level of IL-1β and IL-6 of indicated mouse lung with PAO1 infection (5 × 106 CFU) for 6 h and 24 h. n≥3. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Heatmap showing transcriptome profile correlation values of indicated lung tissue samples before and after PAO1 infection .

Heatmap showing transcriptome profile correlation values of indicated lung tissue samples before and after PAO1 infection.


Histogram of selected differentially expressed genes of LL - 37 + / + mouse lung versus wild type FVB mouse lung prior to infection . Blue bars indicated genes up - regulated in wild type FVB mouse lungs , red bars indicated genes up - regulated in LL - 37 + / + mouse lungs .

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

Histogram of selected differentially expressed genes of LL-37+/+ mouse lung versus wild type FVB mouse lung prior to infection. Blue bars indicated genes up-regulated in wild type FVB mouse lungs, red bars indicated genes up-regulated in LL-37+/+ mouse lungs.


Detection of LL - 37 expression in the engineered mDASCs by

Detection of LL-37 expression in the engineered mDASCs by immunofluorescence


Detection of LL - 37 expression in the engineered mDASCs by real - time quantitative

Detection of LL-37 expression in the engineered mDASCs by real-time quantitative PCR


Detection of LL - 37 expression in the engineered mDASCs by Western

Detection of LL-37 expression in the engineered mDASCs by Western blot


Anti - Krt5 ( red ) and anti - P63 ( green ) immunostaining of WT - and LL - 37 - mDASC colonies . Scale bar , 70 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 O

Anti-Krt5 (red) and anti-P63 (green) immunostaining of WT- and LL-37-mDASC colonies. Scale bar, 70 μm.


Stem cell colony - forming efficiency of WT - and LL - 37 - mDASCs during 5 serial passages . n = 6 .

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

Stem cell colony-forming efficiency of WT- and LL-37-mDASCs during 5 serial passages. n=6.


Representative 3D organoid culture of mDASCs with expression of type I alveolar cell markers ( Aqp5 and Pdpn ) . Left panels , bright field imaging of 3D organoids . Right panels , immunofluorescence of organoid sections . Scale bar , 20 μm .

Representative 3D organoid culture of mDASCs with expression of type I alveolar cell markers (Aqp5 and Pdpn). Left panels, bright field imaging of 3D organoids. Right panels, immunofluorescence of organoid sections. Scale bar, 20 μm.


Co - culture of bacteria with DASC shows the antimicrobial effects in dose - dependent manner . Initial additions of PAO1 were 0 . 1 × , 0 . 5 × and 1 × 104 CFU , respectively . Coculture duration , 6h . n = 4 . Error bars , S . E . M . MOI , Multiplicity of infection .

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

Co-culture of bacteria with DASC shows the antimicrobial effects in dose-dependent manner. Initial additions of PAO1 were 0.1×, 0.5× and 1 × 104 CFU, respectively. Coculture duration, 6h. n=4. Error bars, S.E.M. MOI, Multiplicity of infection.


Co - culture of bacteria with DASC shows antimicrobial effects in time - dependent manner . Initial additions of PAO1 was 1 × 104 CFU . MOI = 1 . n = 3 . Error bars , S . E . M .

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

Co-culture of bacteria with DASC shows antimicrobial effects in time-dependent manner. Initial additions of PAO1 was 1 × 104 CFU. MOI=1. n=3. Error bars, S.E.M.


Preincubation of cells with anti - LL - 37 antibody , but not IgG control , significantly reduced anti - PAO1 and anti - E . coli ( J ) effects of LL - 37 - mDASCs . Initial dose of bacteria was 103 CFU .

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

Preincubation of cells with anti-LL-37 antibody, but not IgG control, significantly reduced anti-PAO1 and anti-E.coli (J) effects of LL-37-mDASCs. Initial dose of bacteria was 103 CFU.


Bright field and direct fluorescence images of mouse lungs following transplantation of 1 × 106 GFP - labeled WT - mDASCs ( WT - Lung ) or LL - 37 - mDASCs ( LL - 37 - Lung ) on indicated days . Scale bar , 1000

Bright field and direct fluorescence images of mouse lungs following transplantation of 1 × 106 GFP-labeled WT-mDASCs (WT-Lung) or LL-37-mDASCs (LL-37-Lung) on indicated days. Scale bar, 1000 μm


Engraftment ratio of transplanted cells in mouse lungs on indicated days . n = 3 . Error bars , S . E . M . * P = 0 . 0474 .

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

Engraftment ratio of transplanted cells in mouse lungs on indicated days. n=3. Error bars, S.E.M. *P=0.0474.


Morphology of engrafted GFP - labeled cells in lung parenchyma by direct fluorescence . Blue color indicates nucleus DAPI staining . Scale bar , 200 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

Morphology of engrafted GFP-labeled cells in lung parenchyma by direct fluorescence. Blue color indicates nucleus DAPI staining. Scale bar, 200 μm.


Anti - Ki67 immunofluorescence of engrafted GFP - labeled WT - and LL - 37 - mDASCs in lung parenchyma 21 days after transplantation . Scale bar , 50 μm .

Anti-Ki67 immunofluorescence of engrafted GFP-labeled WT- and LL-37-mDASCs in lung parenchyma 21 days after transplantation. Scale bar, 50 μm.


Distribution of engrafted GFP - labeled cells in lung parenchyma by immunofluorescence 7 days after transplantation . WT - Lung : WT - mDASCs engrafted ; LL - 37 - Lung : LL - 37 - mDASCs engrafted . Scale bar , 200 μm . Arrows showed the representative cells with overlapping fluorescence of GFP and LL - 37 .

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

Distribution of engrafted GFP-labeled cells in lung parenchyma by immunofluorescence 7 days after transplantation. WT-Lung: WT-mDASCs engrafted; LL-37-Lung: LL-37-mDASCs engrafted. Scale bar, 200 μm. Arrows showed the representative cells with overlapping fluorescence of GFP and LL-37.


Intratracheal instillation of equal amount of PAO1 ( 5 × 106 CFU per mouse ) into WT - Lung ( WT - mDASCs engrafted ) and LL - 37 - Lung ( LL - 37 - mDASCs engrafted ) followed by bacterial CFU analysis in whole lung homogenates 6 , 24 and 48 hours after infection . n = 3 . Error bars , S . E . M . Intratracheal instillation of equal amount of PAO1 into WT - Lung and LL - 37 - Lung followed by bacterial CFU analysis in BAL fluid 6 , 24 and 48 hours after infection . n = 3 . Error bars , S . E . M .

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

Intratracheal instillation of equal amount of PAO1(5 ×106 CFU per mouse) into WT-Lung (WT-mDASCs engrafted) and LL-37-Lung (LL-37-mDASCs engrafted) followed by bacterial CFU analysis in whole lung homogenates 6, 24 and 48 hours after infection. n=3. Error bars, S.E.M. Intratracheal instillation of equal amount of PAO1 into WT-Lung and LL-37-Lung followed by bacterial CFU analysis in BAL fluid 6, 24 and 48 hours after infection. n=3. Error bars, S.E.M.


Arterial blood gas analysis of mice with WT - Lung and LL - 37 - Lung following PAO1 infection . n = 6 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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

Arterial blood gas analysis of mice with WT-Lung and LL-37-Lung following PAO1 infection. n=6. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


H & E staining showing histology of injured lung infected by PAO1 with WT - mDASCs or LL - 37 - mDASCs transplantation . Scale bar , 100 μm . Histopathological examination according to the lung injury scoring system based on blinded expert judgement . n = 5 mice per group . Error bars , S . E . M .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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&E staining showing histology of injured lung infected by PAO1 with WT-mDASCs or LL-37-mDASCs transplantation. Scale bar, 100 μm. Histopathological examination according to the lung injury scoring system based on blinded expert judgement. n=5 mice per group. Error bars, S.E.M.


CD68 immunochemistry ( brown ) in infected lung with WT - mDASCs or LL - 37 - mDASCs transplantation . Scale bar , 50 μm . Quantification of brown - stained ( CD68 + ) area by Image J software . n = 5 . Error bars , S . E . M .

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

CD68 immunochemistry (brown) in infected lung with WT-mDASCs or LL-37-mDASCs transplantation. Scale bar, 50 μm. Quantification of brown-stained (CD68+) area by Image J software. n=5. Error bars, S.E.M.


Gene expression level of indicated pro - inflammatory cytokines of lung infected by PAO1 with WT - mDASCs or LL - 37 - mDASCs transplantation . n = 3 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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

Gene expression level of indicated pro-inflammatory cytokines of lung infected by PAO1 with WT-mDASCs or LL-37-mDASCs transplantation. n=3. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Anti - Krt5 ( red ) and anti - P63 ( green ) immunofluorescence of isolated hDASCs . Scale bar , 90 μm .

Anti-Krt5 (red) and anti-P63 (green) immunofluorescence of isolated hDASCs. Scale bar, 90 μm.


The mRNA expression levels of LL - 37 was measured by qPCR for WT - and LL - 37 - hDASCs . n = 5 . Error bars , S . E . M .

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

The mRNA expression levels of LL-37 was measured by qPCR for WT- and LL-37-hDASCs. n=5. Error bars, S.E.M.


H & E staining of native rat lung tissue and decellularized scaffold showed the absence of nuclei following decellularization . Scale bar , 40 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

H&E staining of native rat lung tissue and decellularized scaffold showed the absence of nuclei following decellularization. Scale bar, 40 μm.


Ex vivo biomimetic culture of LL - 37 - hDASCs recellularized lungs with constant media perfusion .

Ex vivo biomimetic culture of LL-37-hDASCs recellularized lungs with constant media perfusion.


Direct fluorescence image showing GFP - labeled LL - 37 - hDASCs before ( left ) and 7 days after ( right ) being engrafted onto the decellularized scaffold . Scale bar , 100 μm ( right panel ) and 1000 μm ( right panel )

Direct fluorescence image showing GFP-labeled LL-37-hDASCs before (left) and 7 days after (right) being engrafted onto the decellularized scaffold. Scale bar, 100 μm (right panel) and 1000 μm (right panel)


Immunofluorescence of recellularized lung with indicated antibodies . Blue color indicates nucleus DAPI staining . Scale bar , 50 μm .

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

Immunofluorescence of recellularized lung with indicated antibodies. Blue color indicates nucleus DAPI staining. Scale bar, 50 μm.


The recellularized lung by LL - 37 - hDASCs displayed growth inhibitory effect on PAO1 and E . coli . Initial dose of bacteria was 2 × 104 CFU . Culture duration , 24h . n = 6 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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

The recellularized lung by LL-37-hDASCs displayed growth inhibitory effect on PAO1 and E.coli. Initial dose of bacteria was 2× 104 CFU. Culture duration, 24h. n=6. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


A , B . A missense mutation in SPOP gene ( Q165P , T > G ) was identified in a PCa patient . Exome sequencing revealed that the primary tumor contained a heterozygous Q165P mutation ( A ) whereas the liver metastasis harbored a homozygous Q165P mutation ( B ) .

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

A, B. A missense mutation in SPOP gene (Q165P, T >G) was identified in a PCa patient. Exome sequencing revealed that the primary tumor contained a heterozygous Q165P mutation (A) whereas the liver metastasis harbored a homozygous Q165P mutation (B).


D , E . IHC analysis of BRD4 protein expression in SPOP wild type ( WT ) and mutated ( MUT ) PCa patient samples . The representative images of BRD4 IHC staining in both SPOP WT and MUT PCa patients are shown in ( D ) and the quantified IHC data are shown in ( E ) . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields . The red dot in ( E ) indicates SPOP Q165P sample . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * P < 0 . 01 . See Appendix Table S4 for the detailed comparison , P values and sample number ( n ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E. IHC analysis of BRD4 protein expression in SPOP wild type (WT) and mutated (MUT) PCa patient samples. The representative images of BRD4 IHC staining in both SPOP WT and MUT PCa patients are shown in (D) and the quantified IHC data are shown in (E). Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields. The red dot in (E) indicates SPOP Q165P sample. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; ** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).


F , G . IHC analysis of AR protein expression in SPOP WT and mutated PCa patient samples . The representative images of AR IHC staining in both SPOP WT and mutant PCa patients are shown in ( F ) and the quantified IHC data are shown in ( G ) . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields . The green dot indicates SPOP Q165P sample . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * * P < 0 . 01 . See Appendix Table S4 for the detailed comparison , P values and sample number ( n ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. IHC analysis of AR protein expression in SPOP WT and mutated PCa patient samples. The representative images of AR IHC staining in both SPOP WT and mutant PCa patients are shown in (F) and the quantified IHC data are shown in (G). Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields. The green dot indicates SPOP Q165P sample. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; *** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).


LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP ( A )

LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP (A)


LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the BRD4 ubiquitination ( B ) .

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

LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the BRD4 ubiquitination (B).


C . C4 - 2 cells were stably infected with the lenti - virus expressing empty vector ( EV ) or SPOP mutants and harvested for western blot analysis with the indicated antibodies .

O O O O O O O O O O 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. C4-2 cells were stably infected with the lenti-virus expressing empty vector (EV) or SPOP mutants and harvested for western blot analysis with the indicated antibodies.


D . C4 - 2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for western blot analysis with the indicated antibodies . T1 : tumor 1 ; T2 : tumor 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 O O O

D. C4-2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for western blot analysis with the indicated antibodies. T1: tumor 1; T2: tumor 2.


E . Representative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields .

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

E. Representative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors. Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields.


Co - IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant ( F )

Co-IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant (F)


Co - IP assay was performed to detect the interaction between CULLIN3 with SPOP WT or Q165P ( G ) .

Co-IP assay was performed to detect the interaction between CULLIN3 with SPOP WT or Q165P (G).


A . DU145 PCa cells infected with virus expressing EV or SPOP Q165P were harvested for western blot analysis .

A. DU145 PCa cells infected with virus expressing EV or SPOP Q165P were harvested for western blot analysis.


B . MTS was performed to compare the growth between EV and Q165P DU145 cells after the treatment with indicated inhibitors ( 2 µM of JQ1 , 2 µM of CPI - 637 , 2 µM NEO2734 ) for 72 h . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 .

O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

B. MTS was performed to compare the growth between EV and Q165P DU145 cells after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.


Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP Q165P DU145 cells . The survival curve showed IC50 for SPOP Q165P ( 0 . 69 µM ) and EV cells ( 1 . 08 µM ) ( C ) .

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

Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP Q165P DU145 cells. The survival curve showed IC50 for SPOP Q165P (0.69 µM) and EV cells (1.08 µM) (C).


DU145 cells expressing lenti - EV or lenti - HA - SPOP - Q165 constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvest . The number of colonies with more than 50 cells was counted . Representative images of colonies are shown in ( D ) with quantitative data shown in ( E ) . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 .

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

DU145 cells expressing lenti-EV or lenti-HA-SPOP-Q165 constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvest. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantitative data shown in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01.


F . DU145 cells were treated with the indicated inhibitors and harvested for ChIP with BRD4 antibody . The enrichment of BRD4 at c - MYC enhancer and RAC1 promoter was analyzed using qPCR . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DU145 cells were treated with the indicated inhibitors and harvested for ChIP with BRD4 antibody. The enrichment of BRD4 at c-MYC enhancer and RAC1 promoter was analyzed using qPCR. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01.


G . DU145 cells were treated with the indicated inhibitors and harvested for ChIP with Ac - H3 antibody . The level of Ac - H3 at c - MYC enhancer and RAC1 promoter is analyzed using qPCR . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DU145 cells were treated with the indicated inhibitors and harvested for ChIP with Ac-H3 antibody. The level of Ac-H3 at c-MYC enhancer and RAC1 promoter is analyzed using qPCR. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; ** P < 0.01.


B , C . SCID male mice with PDX tumors were treated with vehicle ( 40 % polyethylene glycol ) , JQ1 ( 50 mg / kg ) , CPI - 637 ( 30 mg / kg ) , combination of JQ1 ( 50 mg / kg ) and CPI - 637 ( 30 mg / kg ) or NEO2734 ( 30 mg / kg ) five days a week for three consecutive weeks . Tumors isolated from mice at day 21 of drug treatment were photographed ( B ) and tumor growth are shown in ( C ) . All data shown are means ± SEM . The P value comparing the tumor volume at day 21 post - treatment was calculated by the unpaired two - tailed Student ' s t - test ; * * * P < 0 . 001 .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SCID male mice with PDX tumors were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg), CPI-637 (30 mg/kg), combination of JQ1 (50 mg/kg) and CPI-637 (30 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of drug treatment were photographed (B) and tumor growth are shown in (C). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; *** P < 0.001.


PDX tumors in each treatment group were harvested for protein For western blot analysis , three tumors each group ( n = 3 ) were used for protein extraction and mixed with equal ration to detect the indicated antibodies in ( D ) .

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

PDX tumors in each treatment group were harvested for protein For western blot analysis, three tumors each group (n = 3) were used for protein extraction and mixed with equal ration to detect the indicated antibodies in (D).


PDX tumors in each treatment group were harvested for RNA extraction . For RT - qPCR , three tumors each group ( n = 3 ) were extracted RNA for RT - qPCR in ( E ) . Data are shown as means ± SEM . The P value comparing vehicle with each treatment group was calculated by unpaired two - tailed Student ' s t - test and indicated in the graph .

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

PDX tumors in each treatment group were harvested for RNA extraction. For RT-qPCR, three tumors each group (n = 3) were extracted RNA for RT-qPCR in (E). Data are shown as means ± SEM. The P value comparing vehicle with each treatment group was calculated by unpaired two-tailed Student's t-test and indicated in the graph.


Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were harvested for western blot analysis with the indicated antibodies ( A ) Asterisk , SRC3 at expected molecular mass .

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

Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were harvested for western blot analysis with the indicated antibodies (A) Asterisk, SRC3 at expected molecular mass.


Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were harvested for IFC with p - AKT - S473 antibody ( B ) . E - cadherin antibody was used to indicate the cell membrane ( red ) and DAPI for nucleus ( blue ) . Scale bars : 25 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were harvested for IFC with p-AKT-S473 antibody (B). E-cadherin antibody was used to indicate the cell membrane (red) and DAPI for nucleus (blue). Scale bars: 25 µm.


Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were cultured for five days , followed by the treatment with JQ1 ( 2 µM ) , CPI - 637 ( 2 µM ) , JQ1 ( 2 µM ) + CPI - 637 ( 2 µM ) or NEO2734 ( 2 µM ) for five more days . The representative images of organoids after the treatment are shown in ( C )

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

Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. The representative images of organoids after the treatment are shown in (C)


Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were cultured for five days , followed by the treatment with JQ1 ( 2 µM ) , CPI - 637 ( 2 µM ) , JQ1 ( 2 µM ) + CPI - 637 ( 2 µM ) or NEO2734 ( 2 µM ) for five more days . the quantified data of the organoid diameter are shown in ( D ) . Scale bars : 25 µm . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. the quantified data of the organoid diameter are shown in (D). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.


E , F . 313HR ( SPOP WT ) and 573R ( Q165P ) organoids were cultured for 5 days , followed by the treatment with the indicated inhibitors for 5 days . The organoids were stained with Caspase - 3 antibody ( green ) to detect apoptotic cells . E - cadherin antibody staining was used to define the cell membrane ( red ) and the nucleus was counterstained with DAPI ( blue ) . The representative images of cleaved Caspase - 3 IFC staining are shown in ( E ) . Scale bars : 25 µm . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F. 313HR (SPOP WT) and 573R (Q165P) organoids were cultured for 5 days, followed by the treatment with the indicated inhibitors for 5 days. The organoids were stained with Caspase-3 antibody (green) to detect apoptotic cells. E-cadherin antibody staining was used to define the cell membrane (red) and the nucleus was counterstained with DAPI (blue). The representative images of cleaved Caspase-3 IFC staining are shown in (E). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.


A . C4 - 2 PCa cells infected with virus expressing EV , SPOP Q165P or F133V were harvested for western blot analysis .

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. C4-2 PCa cells infected with virus expressing EV, SPOP Q165P or F133V were harvested for western blot analysis.


B MTS was performed to compare the cell growth between EV , Q165P , F133V C4 - 2 groups after the treatment with indicated inhibitors ( 2 µM of JQ1 , 2 µM of CPI - 637 , 2 µM NEO2734 ) for 72 h . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

B MTS was performed to compare the cell growth between EV, Q165P, F133V C4-2 groups after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.


C . Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4 - 2 cells . The survival curve showed IC50 for EV ( 1 . 01 µM ) , SPOP Q165P ( 0 . 621 µM ) and SPOP F133V cells ( 0 . 395 µM ) .

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

C. Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4-2 cells. The survival curve showed IC50 for EV (1.01 µM), SPOP Q165P (0.621 µM) and SPOP F133V cells (0.395 µM).


D , E . C4 - 2 cells expressing EV , Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting . The number of colonies with more than 50 cells was counted . Representative images of colonies are shown in ( D ) with quantification data in ( E ) . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 .

D, E. C4-2 cells expressing EV, Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantification data in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.


F - H . A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration ( F ) . When the tumor reached 100 mm3 , mice were treated with vehicle ( 40 % polyethylene glycol ) , JQ1 ( 50 mg / kg ) or NEO2734 ( 30 mg / kg ) five days a week for three consecutive weeks . Tumors isolated from mice at day 21 of treatment were photographed ( G ) and tumor growth are shown in ( H ) . All data shown are means ± SEM . The P value comparing the tumor volume at day 21 post - treatment was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - H. A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration (F). When the tumor reached 100 mm3, mice were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of treatment were photographed (G) and tumor growth are shown in (H). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01; *** P < 0.001.


Expression of YAP / TAZ and a smaller protein ( asterisk ) in different cell lines , protein expression was determined by immunoblotting ( IB ) .

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

Expression of YAP/TAZ and a smaller protein (asterisk) in different cell lines, protein expression was determined by immunoblotting (IB).


The shRNA targeting TAZ , but not YAP , knocked down the expression of the smaller protein ( asterisk ) .

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

The shRNA targeting TAZ, but not YAP, knocked down the expression of the smaller protein (asterisk).


Antibodies targeting C - terminus YAP / TAZ , such as TAZ ( CST ) and YAP / TAZ , effectively pulled down the smaller protein ( asterisk , dubbed as cTAZ hereafter ) in RKO cells in an immunoprecipitation ( IP ) assay . cTAZ was not recognized by TAZ ( SA ) , an antibody targeting N - terminus of TAZ .

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

Antibodies targeting C-terminus YAP/TAZ, such as TAZ (CST) and YAP/TAZ, effectively pulled down the smaller protein (asterisk, dubbed as cTAZ hereafter) in RKO cells in an immunoprecipitation (IP) assay. cTAZ was not recognized by TAZ (SA), an antibody targeting N-terminus of TAZ.


Exogenous TAZ / YAP was not processed proteolytically to cTAZ . RKO cells were transfected with the indicated plasmids expressing C - terminal HA - tagged TAZ or YAP .

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

Exogenous TAZ/YAP was not processed proteolytically to cTAZ. RKO cells were transfected with the indicated plasmids expressing C-terminal HA-tagged TAZ or YAP.


RKO , but not HEK293A cells , expressed both full - length TAZ and cTAZ transcripts . RT PCR primers targeting different

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

RKO, but not HEK293A cells, expressed both full-length TAZ and cTAZ transcripts. RT PCR primers targeting different regions


Alignment of TAZ and cTAZ protein sequences . cTAZ protein sequence was derived from the Sanger sequencing results of the short PCR product ( RKO , F2 : R1 )

Alignment of TAZ and cTAZ protein sequences. cTAZ protein sequence was derived from the Sanger sequencing results of the short PCR product (RKO, F2:R1)


Knock out of cTAZ in RKO cells using CRISPR / Cas9 technology . Monoclones ( # 3 and # 14 ) were selected .

Knock out of cTAZ in RKO cells using CRISPR/Cas9 technology. Monoclones (#3 and #14) were selected.


Cluster analysis of RNA expression of WT , cTAZ - / - , and cTAZ - / - / GFP - cTAZ put - back RKO cells . Genes down - and up - regulated more than 2 folds ( P < 0 . 05 ) in cTAZ KO cells were included in cluster analysis . Green and red indicate down - and up - regulated genes respectively .

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

Cluster analysis of RNA expression of WT, cTAZ-/-, and cTAZ-/- /GFP-cTAZ put-back RKO cells. Genes down- and up-regulated more than 2 folds (P < 0.05) in cTAZ KO cells were included in cluster analysis. Green and red indicate down- and up-regulated genes respectively.


cTAZ suppressed expression of ISGs . RNA levels of selected ISGs in WT , cTAZ - / - , cTAZ - / - / GFP - cTAZ RKO cells were quantified by qPCR . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - test .

cTAZ suppressed expression of ISGs. RNA levels of selected ISGs in WT, cTAZ-/-, cTAZ-/-/GFP-cTAZ RKO cells were quantified by qPCR. Error bars indicate SD, n =3. **P<0.01; ***P<0.001; Student's t-test.


cTAZ repressed 5 × ISRE - luciferase activity induced by IFN - α . HEK293A cells were transfected with 5 × ISRE - luciferase reporter along with the indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 12 hours . The expression of ectopic genes was determined by IB . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; one - way ANOVA test was used for statistical analysis .

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

cTAZ repressed 5×ISRE-luciferase activity induced by IFN-α. HEK293A cells were transfected with 5×ISRE-luciferase reporter along with the indicated plasmids, treated with or without IFN-α (50 ng/ml) for 12 hours. The expression of ectopic genes was determined by IB. Error bars indicate SD, n =3. ***P<0.001; one-way ANOVA test was used for statistical analysis.