Fig. 6
Roles of KLF5 acetylation by GCN5 in C5a-induced GDF15 gene transcription, expression and A549 cell proliferation. a Schematic drawing of conserved domains in human GCN5 protein. The GNAT-SF ___domain has HAT activity. b A549 cells were overexpressed with GCN5 and flag-KLF5, and the purified KLF5 was analyzed by mass spectrometry. It exhibited that lysine 335 (K335) and lysine 391 (K391) of KLF5 protein were acetylated. Alignment of KLF5 amino acids sequence among different species showed K335 and K391 are highly conserved. c, d A549 cells transfected with the vectors expressing GCN5-WT, △GCN5 (GCN5 lack of GNAT-SF), flag-tagged KLF5-WT, KLF5-335R, KLF5-391R (lysine to arginine mutant), and KLF5-335/391R (double mutation of lysine 335 and 391). The KLF5 acetylation level detected by IP with abs against KLF5 or flag and IB with anti-Ac-K notably decreased in △GCN5 + C5a group compared with GCN5-WT + C5a (c), and was markedly lower in KLF5–K335R + C5a, KLF5-391R + C5a and KLF5–K335/391R + C5a groups than in KLF5-WT + C5a group (d), particularly more significantly in KLF5–K335/391R + C5a group (*P < 0.05, **P < 0.01). e Luciferase reporter analysis displayed that GDF15 promoter (FL) activity in △GCN5 + C5a and KLF5–K335/391R + C5a groups was decreased (*P < 0.05 vs. GCN5-WT + C5a; △△P < 0.01 vs. KLF5-WT + C5a). f, g Real-time PCR (f) and IB (g) assays manifested that GDF15 mRNA and protein in △GCN5 + C5a and KLF5–K335/391R + C5a groups were diminished (*P < 0.05 vs. GCN5-WT + C5a; △△P < 0.01 vs. KLF5-WT + C5a). h–j CCK8 (h), cell counting (i), and colony formation (j) assays showed the proliferation of A549 cells in △GCN5 + C5a and KLF5–K335/391R + C5a groups was lessened (*P < 0.05 vs. GCN5-WT + C5a; △△P < 0.01 vs. KLF5-WT + C5a). Means ± S.E.M. is presented, and results are representative of three independent experiments. Representative photographs are exhibited
