Fig. 4.

Inefficient phosphorylation of H2AX is independent of DNA methylation levels in mouse and human fibroblasts. (A) Western blots probed with anti-γH2AX and anti-H2AX antibodies before and after irradiation of wild-type (WT), Lsh^−/−, p53^−/− and DNA methylation-deficient Dnmt1^−/−; p53^−/− MEFs. Note that Dnmt1^−/−; p53^−/− MEFs show normal levels of γH2AX when compared to controls. (B) Western blot detecting the successful stable KD of LSH (LSH KD) in hTERT-immortalized MRC5 human lung fibroblasts in comparison to non-infected and control cells infected with non-silencing shRNA lentiviruses (Con KD). (C) Quantitative RT-PCR detecting LSH mRNA levels relative to GAPDH in control and LSH KD MRC5 cells. (D) The levels of 5-methyl cytosine (5-meC) in LSH KD and control cells were detected by ELISA and quantified as % 5-meC detected in non-infected MRC5 cells. (E) MRC5 cells in which LSH was knocked down show reduced γH2AX after irradiation when compared to control cells. All samples were run on the same gel and detected simultaneously. H2AX and H4 serve as loading controls. (F) Quantification of γH2AX signal relative to H2AX from three independent experiments. (G) LSH-deficient MRC5 fibroblasts show reduced survival in colony formation assays in comparison to control cells expressing non-silencing shRNA. The error bars in C, D, F and G represent S.D.