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. 2017 Aug 11;8:16016. doi: 10.1038/ncomms16016

Figure 8. SRSF1-triggered activation of KLF5 requires EGR1 binding to Δ133p53.

Figure 8

(a) Co-immunoprecipitation (IP) assay identified interaction between Δ133p53 and EGR1. Cell lysates from HASMCs infected with Ad-β-gal or Ad-Δ133p53-myc were immunoprecipitated with antibody to myc (left) or EGR1 (right) and detected with western blots. Input lysates were loaded as control; n=5 per group. (b) Chromatin immunoprecipitation (ChIP) assays were performed to detect the recruitment of EGR1 protein to the promoter of KLF5 after overexpression of Δ133p53. HASMCs were infected with Ad-β-gal or Ad-Δ133p53-myc. Crosslinked DNA–protein lysates are immunoprecipitated with antibody to EGR1. The fragments containing consensus EGR1-binding motifs are PCR amplified from both samples. Four distinct EGR1-binding positions (P1, P2, P3 and P4) in KLF5 are shown. n=9 per group. (c) Representative western blots and averaged data showing the EGR1 levels in HASMCs infected with scrambled or two sets of EGR1 siRNAs (EGR1 si1 and EGR1 si2). (d) ChIP assays were performed in HASMCs infected with Ad-β-gal or Ad-Δ133p53-myc in the presence or absence of EGR1 siRNA1 (EGR1 si); n=9 per group. Crosslinked DNA–protein lysates from infected cells are immunoprecipitated with EGR1 antibody. The P1 and P2 promoter fragments (same as Fig. 7b) containing EGR1-binding motifs are PCR amplified from both samples. (e) Representative western blots and averaged data showing KLF5, p21 and PCNA levels in HASMCs transfected with EGR1 siRNAs with or without Ad-SRSF1 overexpression; n=7 per group. (f) Representative western blots and averaged data showing KLF5, p21 and PCNA levels in HASMCs transfected with EGR1 siRNAs with or without Ad-Δ133p53 overexpression; n=9 per group. (g,h) The proliferation enhancement induced by SRSF1 (g) or Δ133p53 (h) was attenuated by EGR1 siRNAs; n=12 per group. Scr indicates scrambled siRNA control. *P<0.05, **P<0.01, NS, not significant; Student’s t-test (b) or one-way ANOVA (d–h). Data are mean±s.e.m. of four (b,d) or five (e–h) independent experiments.