Fig 2: KRASMut lysine 104 is acetylated by the p300 acetyltransferase.A HEK293T cells were transfected with KRASWT, Flag-E.V., and Flag-p300 plasmids. Cell lysates were immunoprecipitated with IgG and an anti-KRAS antibody and then immunoblotted with anti-acetyl-lysine, anti-Flag, and anti-KRAS antibodies. B, C Myc-His-KRASG12C, Flag-SBP-E.V., Flag-SBP-p300, siCon, and/or sip300 were overexpressed in HEK293T cells; after 48 h, cell lysates were separated into nuclear and cytoplasmic fractions, which were subjected to immunoprecipitation with an anti-KRAS antibody and immunoblotting with anti-acetylated lysine and anti-KRAS antibodies. TATA-binding protein was used as a nuclear marker, and tubulin was used as a cytoplasmic marker to verify that nuclear separation was successful. D HEK293T cells were transfected with GFP-E.V., GFP-KRAS (all lysine residues between a.a. 101 and 147 except for K104 mutated to arginine), Flag-SBP-E.V., Flag-SBP-p300, siCon, and/or sip300. Protein lysates were immunoprecipitated with an anti-GFP antibody and then immunoblotted with anti-acetylated lysine, anti-KRAS, and anti-GFP antibodies. E The recombinant KRASG12C protein and the p300 C.D. (catalytic domain, aa 1284–1674, 45.1 kDa) were incubated in the acetylation assay reaction mixture at 32 °C for 4 h, and lysine-acetylated peptides were then identified using an anti-acetyl-lysine antibody. F Lysine-acetylated KRASG12C peptides were also incubated with recombinant SIRT1 protein in the deacetylation assay reaction mixture for 4 h. Deacetylation was quantified using an anti-acetyl-lysine antibody.

Fig 3: SIRT1 upregulation is mediated by c-Myc downstream of KRAS.A HEK293T cells were transfected with pcDNA and KRASG12C plasmids (2 μg). H460 cells were transfected with siCon and siKRAS (80 nM). The cells were harvested with lysis buffer and subjected to western blotting. B, C KRASMut cells (H358, NCIH23, SKLU-1, SW900, A427, H727), KRASWT cells, and KRASG12C cells (H1299G12C) were transfected with siCon, c-Myc specific siRNA (80 nM), pcDNA, or c-Myc plasmid (2 μg) for 48 h, and the levels of the KRAS downstream effectors c-Myc and SIRT1 were measured. D H358 cells were transfected with KRASG12C and siCon or sic-Myc, and cell extracts were immunoprecipitated with an anti-KRAS antibody and immunoblotted with anti-SIRT1, anti-c-Myc, and anti-KRAS antibodies. E Chromatin immunoprecipitation-qPCR analysis of KRAS, SIRT1, and SIRT2 was performed in H358 cells transfected with siCon or siKRAS (80 nM) for 48 h and then immunoprecipitated using an anti-c-Myc antibody or mouse IgG as a negative control. The relative enrichment was calculated by normalizing the qPCR signals. The data are plotted as the mean values determined from at least two independent chromatin immunoprecipitation assays and three independent amplification reactions. Student’s t test, mean ± SD; n = 6; *p < 0.05. F H358 cells were transfected with siCon and siKRAS (80 nM) for 48 h and then fixed after 4 h. c-Myc expression was detected with an RFP emission filter, and SIRT1 expression was detected with a GFP emission filter.

Fig 4: KRASMut-induced SIRT1 rebinds to KRASMut and increases KRAS activity via deacetylation.A HEK293T cells were transfected with KRASG12C and SIRT1 plasmids (4 μg), and cell extracts were immunoprecipitated with anti-KRAS and anti-SIRT1 antibodies and immunoblotted with the reciprocal antibody. B, C Plasmids (pcDNA, KRASG12C, and SIRT1 each 4 μg) and siRNAs (siCon and siSIRT1, each 80 nM) were transfected into HEK293T cells. Cell extracts were immunoprecipitated with an anti-KRAS antibody and Raf-1 agarose beads and analyzed using anti-acetylated lysine, anti-SIRT1, anti-KRAS, and anti-KRAS-GTP antibodies. D Normal lung epithelial cell, fibroblast, and KRAS Mut cell lysates were immunoprecipitated with an anti-KRAS antibody and immunoblotted with anti-acetyl-lysine and anti-KRAS antibodies.