Figure 4: The KLF5/BAP1/HCF-1 complex promotes cell cycle progression through p27.

(a) Exogenous KLF5 interacted with BAP1, HCF-1 and OGT. KLF5-3 × Flag and BAP1 were co-expressed in HEK293FT cells. When KLF5 was immunoprecipitated with anti-Flag-M2 beads, exogenous BAP1 and endogenous HCF-1 and OGT co-immunoprecipitated. (b) Endogenous KLF5, BAP1, HCF-1 and OGT proteins are in the same protein complex. The HCC1806 cell lysates were immunoprecipitated with the anti-BAP1 antibody. Endogenous KLF5, HCF-1 and OGT proteins co-immunoprecipitated. The c-Myc protein was not in the protein complex. Immunoglobulin (Ig)G was used as the negative control. (c) Knockdown of each member of the KLF5/BAP1/HCF-1 complex in HCC1806 cells upregulated p27 protein levels and downregulated FGF-BP protein levels. (d) Knockdown of KLF5, BAP1 and HCF-1, but not OGT, upregulated p27 mRNA levels in HCC1806 cells as measured by RT–quantitative (q)PCR (n=3). The expression levels of p27 were normalized to β-actin (mean±s.d. from three experiments in triplicate). Every experimental group (KLF5si, BAP1si, HCF-1si and OGTsi) was compared with the Lucsi group, *P<0.05, t-test. (e) KLF5 binds to the p27 gene promoter as determined by ChIP assays in HCC1806 cells. Either IgG- or ChIP-grade anti-KLF5 antibody was used for ChIP. ChIP signals were determined by qPCR and calculated as percentage IP DNA/input DNA (mean±s.d. from three experiments). IgG was served as a negative control. The KLF5 group was compared with the IgG group. *P<0.05, t-test. (f) BAP1 binds to the p27 gene promoter as determined by ChIP assays in HCC1806 cells (mean±s.d. from three experiments). *P<0.05; NS, not significant, t-test. (g) Knockdown of each member of the KLF5/BAP1/HCF-1 complex in HCC1806 by siRNA for 48 h decreased G1/S cell cycle progression (n=3). Data were presented as percentage G1, S or G2/M phase (mean±s.d. from three experiments in duplicate). Every experimental group (KLF5si, BAP1si, HCF-1si and OGTsi) was compared with the Lucsi group, **P<0.01, t-test. (h) Knockdown of p27 in combination with KLF5, BAP1, HCF-1 or OGT knockdown in HCC1806 cells. The protein levels of p27, KLF5, BAP1, HCF-1 and OGT were analysed by WB. (i) The HCC1806 cell cycle G1-phase arrest induced by the knockdown of KLF5, BAP1, HCF-1 or OGT was rescued by the depletion of p27 (n=3). Data are presented as percentage G1, S or G2/M phase (mean±s.d. from three experiments in duplicate).