Figure 3
From: The histone demethylase KDM4B regulates peritoneal seeding of ovarian cancer
KDM4B regulates different pathways in normoxia and hypoxia. (a) Immunoblotting of KDM4B expression, H3K9me3, H3K9me2, H3K9me1, H3K36me3, H3K36me2 and H3K36me1 in SKOV3ip.1 cells transfected with small interfering RNA (siRNA) to KDM4B (4B) or control siRNA (C). Histone H3 serves as control for protein loading. (b) Quantitative real-time PCR (RT–PCR) of KDM4B expression in SKOV3ip.1 cells transfected with siRNA to KDM4B (siK4B) or control siRNA (siCon) and exposed to 21 or 0.5% oxygen for 16 h. (c) Venn diagram showing the overlap between genes downregulated greater than 1.4-fold by siK4B in normoxia (top circle) and hypoxia (bottom circle). (d) Quantitative RT–PCR validation of selected metastasis-associated genes regulated by KDM4B. Data in (b) and (d) represent mean fold change±s.e.m. normalized to 18S rRNA, calculated relative to siCon in 21% O2 (black bars). Results were averaged from three independent experiments, measured in triplicate. Significance of differences was calculated using two-tailed paired Student’s t-test (!P<0.05 for siK4B compared with siCon; #P<0.05 for hypoxia compared with normoxia) or two-way analysis of variance (ANOVA) (*P<0.05 for interactions between oxygen conditions and KDM4B expression). (e) Quantitative RT–PCR measurement of PDGFB, LCN2 and LOX in SKOV3ip.1 cells expressing shRNA to KDM4B (shK-1, dark gray and shK-2, light gray) cultured in the indicated oxygen tensions. Data in (e) represent mean fold change±s.e.m., normalized to 18S rRNA and shGFP control at 21% oxygen (n=5 measured in triplicate). *P<0.05, determined by two-tailed paired Student’s t-test, relative to shGFP control at the respective oxygen tension. (f) Kaplan–Meier progression-free survival plot of LOX expression in all stages of serous epithelial ovarian cancer from data set curated by Gyorffy et al.20 (P<0.05).
