Figure 4
From: The histone demethylase KDM4B regulates peritoneal seeding of ovarian cancer
KDM4B binds regions proximal to target gene promoters and demethylates histone H3K9me3 to regulate expression. (a) Immunoblot measurement of KDM4B and H3K9me3 in SKOV3ip.1 and OVCAR8 cells expressing shRNA targeting KDM4B (shK-1 and shK-2) or GFP (control) in 21, 2 and 0.5% O2. Tubulin and histone H3 serve as loading controls. (b) Map of chromatin immunoprecipitation (ChIP) quantitative PCR (qPCR) primers sets used to measure KDM4B association and histone H3K9 methylation at or near target gene promoters. (c) ChIP assay for KDM4B near promoters of PDGFB, LCN2, LOX and LOXL2 genes in SKOV3ip.1 cells expressing shRNA to KDM4B (shK-2, light gray bars) compared with shGFP control (black bars). Cells were treated with normoxia (21%) or hypoxia (0.5%) for 16 h. Data represent mean fold enrichment±s.e.m. of three independent experiments (n=3) measured in triplicate, normalized input and then to GFP control at 21% O2. (d) ChIP qPCR analysis of H3K9me3 on indicated promoter regions of the PDGFB, LCN2, LOX and LOXL2 genes following shRNA to KDM4B (shK-2, light gray bars) compared with shGFP control (black bars). Data represent the mean of four independent experiments±s.e.m. (n=4) measured in triplicate, and normalized to shGFP control following subtraction of IgG signal and normalization to a gene desert control region as described in the Materials and methods. (e) H3K9me3 ChIP to a gene desert control region. Data represent the mean of four independent experiments±s.e.m. (n=4) measured in triplicate normalized to input. Significance relative to shGFP control was determined using paired Student’s t-test (*P<0.05).
