Fig 2: Analysis of KLF6 acetylation mediated by KAT7 in GMCs induced by sublytic C5b-9. (A) IB analysis of acetylated lysine (Ac-K), KLF6 and KAT7 in renal cortex of SD rats injected intravenously with Thy-1 Ab for the indicated times, assessed after IP with antibody to KLF6 (*P < 0.05, **P < 0.01 vs. 0 h). (B) IB analysis of acetylated lysine (Ac-K) and KLF6 in renal cortex of SD rats injected intravenously with Thy-1 Ab or normal rabbit serum (NS) for 5 h, assessed after IP with antibody to KLF6 (**P < 0.01 vs. NS). (C) IB analysis of acetylated lysine (Ac-K), KLF6 and KAT7 in GMCs stimulated with sublytic C5b-9 for the indicated times, assessed after IP with antibody to KLF6 (**P < 0.01 vs. 0 h). (D) IB analysis of acetylated lysine (Ac-K), KLF6 and KAT7 in GMCs stimulated with sublytic C5b-9, Thy-1 Ab, Thy-1 Ab + HIS, Thy-1 Ab + C6DS, Thy-1 Ab + C6DS + C6, or MEM for 3 h, assessed after IP with antibody to KLF6 (**P < 0.01 vs. Thy-1 Ab, Thy-1 Ab + HIS, Thy-1 Ab + C6DS, and MEM; ??P < 0.01 vs. Thy-1 Ab + C6DS). (E) IB analysis of acetylated lysine (Ac-K), KLF6 and KAT7 in GMCs transfected with pIRES2, pIRES2-KAT7, shCTR or shKAT7 for 48 h and then incubated with sublytic C5b-9 for 3 h, assessed after IP with antibody to KLF6. Data are representative of three independent experiments with similar results or are shown as mean ± SD from three independent experiments.

Fig 3: Role of KLF6 and KAT7 expression in sublytic C5b-9-induced MCP-1 and RANTES expression. GMCs were transfected with control vector (pIRES2), vector encoding KLF6 (pIRES2-KLF6) or KAT7 (pIRES2-KAT7), control shRNA (shCTR), or specific shRNA targeting KLF6 (shKLF6) or KAT7 (shKAT7) for 48 h and then incubated with or without sublytic C5b-9 for 5 h. (A and C) qPCR analysis of MCP-1 (A) and RANTES (C) mRNA in GMCs. (B and D) ELISA of MCP-1 (B) and RANTES (D) in supernatants of GMCs. **P < 0.01 vs. pIRES2; △△P < 0.01 vs. shCTR + sublytic C5b-9. Data are shown as mean ± SD from three independent experiments.

Fig 4: Effect of KLF6 acetylation on MCP-1 and RANTES gene transcription in GMCs exposed to sublytic C5b-9. (A) Mass spectrometry analysis of acetylated lysine residues in KLF6 protein upon sublytic C5b-9 stimulation. (B) Sequence alignment of potential acetylation sites in KLF6. The conserved lysine residues are in bold and underlined. (C) IB analysis of acetylated lysine (Ac-K), KLF6 and KAT7 in GMCs transfected with vector encoding WT or mutant KLF6 (K100R, K213R and K100R/K213R) along with vector encoding KAT7 for 48 h, assessed after IP with antibody to KLF6. (D) IB analysis of acetylated lysine (Ac-K), KLF6 and KAT7 in GMCs transfected with vector encoding WT or mutant KLF6 (K100R and K100Q) along with vector encoding KAT7 for 48 h and then incubated with sublytic C5b-9 for 3 h, assessed after IP with antibody to KLF6. (E) ELISA of MCP-1 and RANTES in supernatants of GMCs transfected with vector encoding WT or mutant KLF6 (K100R and K100Q) along with vector encoding KAT7 for 48 h and then incubated with sublytic C5b-9 for 5 h. (F) qPCR analysis of MCP-1 and RANTES mRNA in GMCs transfected with vector encoding WT or mutant KLF6 (K100R and K100Q) along with vector encoding KAT7 for 48 h and then incubated with sublytic C5b-9 for 5 h. (G) Luciferase activity assay of MCP-1 reporter (-1670 to -30 nt) or RANTES reporter (-1744 to -14 nt) in GMCs transfected with vector encoding WT or mutant KLF6 (K100R and K100Q) along with vector encoding KAT7 for 48 h and then incubated with sublytic C5b-9 for 5 h. (H and I) ChIP analysis of KLF6 at MCP-1 promoter (-297 to -123 nt) (H) or RANTES promoter (-343 to -191 nt) (I) in GMCs transfected with vector encoding WT or mutant KLF6 (K100R and K100Q) along with vector encoding KAT7 for 48 h and then incubated with sublytic C5b-9 for 5 h. **P < 0.01 vs. KLF6 WT + KAT7 + sublytic C5b-9. Data are representative of three independent experiments with similar results or are shown as mean ± SD from three independent experiments.

Fig 5: PKD1 interacts with KAT7 in vitro and in vivo.a KAT7 were pulled down by Flag-PKD1. Cellular extracts from HEK293T cells overexpressing FLAG-PKD1 were immunoprecipitated with anti-FLAG antibody and Protein G-Sepharose beads. After extensively washing the beads, immobilized proteins were eluted in SDS–PAGE sample buffer. The eluent was resolved by SDS–PAGE and silver stained. The protein bands were retrieved and analyzed by mass spectrometry. b–e PKD1 interacts with KAT7 in vivo. b HEK293T cells were co-transfected with HA-PKD1 and Flag-KAT7 plasmids. IP assay and subsequent western blotting were performed by using the indicated antibodies in the panel. c HEK293T cells were transfected with Flag-KAT7 and HeLa cells were transfected with Flag-PKD1, IP assay was carried out by using anti-FLAG antibody and followed by western blotting with anti-KAT7 or anti-PKD1 antibody, respectively. d, e Whole-cell lysates from HEK293T, HeLa, or H1299 cells were immunoprecipitated with anti-KAT7 or anti-PKD1 antibodies followed by immunoblotting with the indicated antibodies. f PKD1 interacts with KAT7 in vitro. GST pull-down assay were performed with bacterial expressed GST-KAT7 protein and HEK293T-expressed FLAG-PKD1. g The schematic representation of the key domains within KAT7 was depicted, and GST pull-down assay was performed by incubating GST-KAT7 or its various deletion mutants with HEK293T-expressed HA-PKD1.