Fig. 6: SP1-mediated SAT1 regulation is critical for tumour cell survival under acidosis.
a, Consensus sequence of the SP1 binding motif. b, Putative binding sites of SP1 in the promoter region of the SAT1 gene. c, Fold enrichment of SP1 binding in SAT1 gene promoter in S2-013 cells upon acetate treatment normalized to IgG control (n = 4 from independent biological replicates). d,e, Relative levels of SP1 mRNA (d) and protein (e) in PDAC cells upon acetate treatment (n = 4 from independent biological replicates; representative immunoblot images of two independent experiments). f,g, Relative SP1 and SAT1 mRNA levels in scrambled control (siScr) and SP1 knockdown (siSP1) S2-013 (f) and HPAF-II (g) cells upon acetate treatment (n = 4 from independent biological replicates). h, Immunoblots of SP1 and SAT1 in siScr and siSP1 S2-013 and HPAF-II cells, upon treatment with acetate. Images are representative of two independent experiments. i, Relative SAT1 and SP1 mRNA levels in SP1-overexpressing S2-013 cells upon acetate treatment (n = 4 from independent biological replicates). j, SAT1 and SP1 immunoblots in SP1-overexpressing S2-013 cells upon acetate treatment. Images are representative of two independent experiments. k, Immunoblot of lysine-acetylated proteins in S2-013 and HPAF-II cells treated with 0 and 5 mM acetate for 6 h. Images are representative of two independent experiments. l, Schematic representation of acetyl-lysine-modified proteome analysis of S2-013 cells treated with 5 mM acetate for 6 h. m, Venn diagram showing differentially acetylated proteins in S2-013 cells upon acetate treatment. n, LC–MS/MS analysis of acetylated peptide corresponding to SP1 (n = 2 from independent biological replicates). o, Peptide sequence alignment of SP1 (amino acids 1–31) in various species, highlighting the prospective acetylation site K19. p, Detection and quantitation (n = 5) of HA-tagged SP1 wild-type (WT) or K19R mutant in HEK293T cells treated with acetate, without or with MG132. Images are representative of five independent experiments. q,r, Representative immunofluorescent images (q) and quantitation (r) showing expression of SP1 in acidic tumour regions, as imaged by staining with pHLIP in tumour sections from 20-week-old KPC mice. Scale bars, 100 μm (n = 12 for normal pH, n = 24 for acidic pH). Unpaired, two-tailed t-test, mean ± s.e.m. (d,r); one-way ANOVA with Bonferroni’s post-hoc test, mean ± s.e.m. (c,f,g,i,p).
