FIG 6.

PPM1G functions downstream of NF-κB and Pol II recruitment to gene promoters to induce 7SK snRNP disassembly and Pol II elongation in response to DNA damage. (A) HeLa cells were transduced with lentiviruses (pLKO) inducibly expressing nontarget (NTsh) or PPM1G (PPM1Gsh) shRNAs, induced with IPTG for 3 days, and treated with etoposide. PPM1G KD efficiency, induction of DNA damage (γH2AX), and stability of transcriptional components were evaluated by Western blotting over time. (B) PPM1G KD antagonizes IL-8 gene expression in response to etoposide. The cell lines from panel A were used for total RNA extraction, and IL-8 gene expression in response to etoposide was quantified by qRT-PCR (means ± standard errors of the means; n = 3). The data were normalized to levels of the β-actin transcript. (C) ChIP assay of cell lines from panel A showing levels of NF-κB, PPM1G, Pol II, and 7SK snRNP components (Cdk9, Hexim1, and Larp7) at the IL-8 promoter-proximal region (−10 amplicon) and gene body (+2464 amplicon) in response to a time course of etoposide treatment (means ± standard errors of the means; n = 3). Normal serum (IgG) was used as a negative control to demonstrate the specificity of enrichment with the antibodies used.