Extended Data Fig. 5: Role of ACSS2 in acetate-mediated epigenetic reprogramming, survival under acidosis, and tumor burden.
a, Relative survival of CFPAC-1 and T3M4 cells upon treatment with 20 µM ACSS2 inhibitor, without or with acetate (n = 3 in each group from independent biological replicates). b, Representative immunoblots (of two independent experiments) showing the levels of ACSS2, acetylated histones, and total H3 protein in scrambled control (siScr) and ACSS2 knockdown (siACSS2) CFPAC-1 and T3M4 cells upon treatment with 5 mM acetate. c, Relative survival of siScr and siACSS2 CFPAC-1 and T3M4 cells in the presence and absence of acetate (n = 3 in each group from independent biological replicates). d, Immunoblots (representative of two independent experiments) showing the levels of ACSS2 protein in control (shScr) and ACSS2 knockdown (shACSS2- a and shACSS2-b) S2-013, HPAF-II, and CFPAC-1 cells under acidosis. e, Relative survival of control and ACSS2 knockdown HPAF-II and CFPAC-1 cells in the presence, and absence of acetate (n = 6 for HPAF-II and n = 3 for CFPAC-1 in each group from independent biological replicates). f, Representative immunofluorescent images showing co-expression of stellate cell marker (αSMA) and ACSS2 in tumor sections from athymic-nude mice implanted with shScr or shACSS2 S2-013 cells alone or co-implanted with HPS cells. Scale bar = 250 µm. g, Representative images of tumors excised from athymic-nude mice implanted with shScr or shACSS2 CFPAC-1 cells alone or co-implanted with HPS cells. h, Tumor volumes, upon necropsy, of athymic-nude mice implanted with control and ACSS2 knockdown CFPAC-1 cells alone or co-implanted with HPS cells (n = 7, 8, 8, 7, 9, 9 mice in indicated groups). i,j, Representative IHC images for ACSS2 and Ki-67 staining (j) in tumor sections from athymic-nude implanted with shScr or shACSS2 CFPAC-1 cells alone or co-implanted with HPS cells along with the quantitation of percent positive Ki-67 cells (i). Scale bar = 100 µm. Ki-67 positive cells were counted manually in three different fields from 3 tumor sections of each group (n = 9 in each group). k, Representative IHC staining for cleaved caspase-3 protein in tumor sections from athymic-nude mice implanted with shScr or shACSS2 S2-013 cells alone or co-implanted with HPS cells. Scale bar = 100 µm. l,m, Tumor weights (l) and tumor volumes (m), upon necropsy for athymic-nude mice implanted with shScr or shACSS2 (shACSS2-a and shACSS2-b) S2-013 cells. All groups were compared to shScr cohort. (n = 9 mice in each group). n,o, Representative IHC images for Ki-67 staining (n) in tumor sections from athymic-nude mice implanted with shScr or shACSS2 S2-013 cells along with the quantitation of percent Ki-67 positive cells (o). Scale bar = 100 µm. Ki-67 positive cells were counted manually in three different fields from 3 tumor sections of each group (n = 9 in each group). Two-way ANOVA with Tukey’s post-hoc test; mean ± s.e.m. (a,c,e); one-way ANOVA with Bonferroni’s post-hoc test; mean ± s.e.m. (h,i,l,m,o).
