Extended Data Fig. 4: Effect of acetate treatment on lipid biosynthesis, histone acetylation, and ACSS2 expression in pancreatic cancer cells under acidosis.
a, Relative mRNA expression of SREBF1, ACACA and FASN genes in CFPAC-1 and T3M4 cells upon treatment with 5 mM acetate for 12 hrs (n = 4 in each group from independent biological replicates). b,c, Representative fluorescent images (b) showing Nile red staining in CFPAC-1 and T3M4 cells upon acetate treatment for 96 hrs. The bar charts show relative fluorescence levels of Nile red staining (c). Total lipid fluorescence is normalized to the cell count. Scale bar = 100 µm. (n = 5 in each group from independent biological replicates). d,e, Relative quantification of Nile red fluorescence in PDAC cells treated with acetate for 96 hrs. The cells were cultured in a DMEM medium containing normal and delipidated FBS in a 3:1 (d) or 1:3 (e) ratio. The total Nile red fluorescence was normalized to the cell count (n = 8 in each group from independent biological replicates). f,g, Relative mRNA expression of FASN (f) and SREBF1 (g) genes in PDAC cell lines upon treatment with CM from HPS and CAF-0911 cells for 12 hrs (n = 4 in each group from independent biological replicates). h-k, Representative fluorescent images showing the Nile red (h) or BODIPY (j) staining in PDAC cell lines upon treatment with CM from HPS and CAF-0911 cells for 96 hrs. Scale bar = 100 µm. The bar charts show relative fluorescence levels of Nile red (i) or BODIPY (k) staining. Total lipid fluorescence is normalized to the cell count (n = 6 in each group for ‘i’ and n = 4 in each group for ‘k’ from independent biological replicates). l, Relative survival of CFPAC-1 and T3M4 cells upon treatment with control or 100 µM orlistat, without or with acetate (n = 3 in each group from independent biological replicates). m, Relative survival of CFPAC-1 and T3M4 cells upon treatment with 10 µM C646 inhibitor, without or with acetate (n = 3 in each group from independent biological replicates). n, Relative mRNA expression of ACSS2 in S2-013, HPAF-II, CFPAC-1, and T3M4 cells upon treatment with 5 mM acetate for 12 hrs under normal pH conditions (n = 4 in each group from independent biological replicates). o, Representative immunoblots showing expression of ACSS2 in S2-013, HPAF-II, CFPAC-1, and T3M4 cells upon treatment with 5 mM acetate under normal pH conditions. Representative image of two independent experiments. Unpaired, two-tailed t-test; mean ± s.e.m. (a,n); one-way ANOVA with Bonferroni’s post-hoc test; mean ± s.e.m. (c,d,e,f,g,i,k,); two-way ANOVA with Tukey’s multiple comparisons test; mean ± s.e.m.(l,m).
