Previous work shows that G9a is degraded in response to DNA damage in primary human diploid fibroblasts (31). schematic (b) shows the sequencing results of sgRNA regions. del, deletion; in, insert; nt, nucleotide. ( 100). ( 0.05), suggesting that lack of G9a impaired DNA damage repair (Fig. 1 and and 0.05). Altogether, these data indicate that G9a is involved in DNA damage repair and thus affects cell survival. G9a Is Recruited to Chromatin in Response to DNA Damage. Previous work has shown that G9a is degraded in response to DNA damage in primary human diploid fibroblasts (31). Here, the total G9a protein levels did not 8-Gingerol change in HCT116, Rabbit Polyclonal to GPR133 HeLa, or LoVo cancer cells following DNA damage (Fig. S2and and and and Fig. S3or (Fig. S4and 20). Students test (two-tailed): S211D versus WT, 0.05; S211A versus WT, 0.05. Open in a separate window Fig. S4. Phosphorylation of G9a at Ser211 permits its recruitment to chromatin and leads to increased H3K9me2 levels. This figure is related to Fig. 4. (and Fig. S5and and Fig. S5and 0.05). In addition, the cell-cycle distribution was unaltered, indicating that no specific cell-cycle stage is perturbed following G9a depletion (Fig. S6and and Fig. S6100) from three independent experiments. (band of RPA70 8-Gingerol indicates the exogenous RPA70 and the band indicates the endogenous RPA70. ( 0.05), whereas it had little effect on NHEJ. In addition, G9a knockdown did not 8-Gingerol alter the cell-cycle profiles of DR-U2OS or EJ-U2OS cells (Fig. S7and and test (two-tailed): WT versus pcDNA3.1(+) 0.05; WT versus S211A, 0.05; S211D versus pcDNA3.1(+) 0.05; S211D versus S211A, 0.05. Open in a separate window Fig. S9. The interaction between G9a and RPA is required for DNA damage repair. This figure is related to Fig. 7. (and for 30 s, the cell pellets were washed twice in PBS and then lysed in buffer II (3 mM EDTA, 0.2 mM EGTA, 1% mixture, and 1 mM DTT) for 30 min. After centrifugation at 8-Gingerol 12,000 for 3 min, the supernatant was assumed to contain soluble nucleoproteins (Dt), 8-Gingerol and the pellets were assumed to contain the chromatin fraction (Chr). SI Materials and Methods Cell Culture. Cells were grown in DMEM or McCoys 5A with 10% (vol/vol) FBS and the appropriate amount of penicillin/streptomycin in a 37 C incubator with a humidified, 5% CO2 atmosphere. Plasmids Construction. The G9a full-length gene (isoform a) or fragments were separately subcloned into pEGFP-C1, p3xFLAG-CMV-10, or pGEX-6p1 vectors. CK2 was amplified from a cDNA library of HCT116 cells and cloned into p3xFLAG-CMV-10. RPA32 or RPA70 were separately amplified and cloned into p3xFLAG-CMV-10, pGEX-6p1, or m-Cherry-N1 vectors. G9a or CK2 mutants were generated using a site-directed mutagenesis kit (Stratagene). Transient and stable transfections of these plasmids were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturers protocol. Antibodies. The antibodies used were antiCGFP-tag; antiCFlag-tag (Sigma-Aldrich); antiCGST-tag (Applygen); anti-G9a (for confocal, Cell Signaling Technology; for Western blotting and coimmunoprecipitation, Sigma-Aldrich); antiCpan-serine, antiCpan-methyl, anti-H3K9me2, anti-H3, and anti-RPA32 (for confocal, Abcam); anti-BRCA1, anti-Rad51, anti-actin, antiC-tubulin, anti-p53S15ph, anti-RPA32 (for Western blotting, Santa Cruz); antiCphospho-Histone H2AX (Ser139) (for confocal microscopy, Millipore; for Western blot, Cell Signaling Technology); antiCp-RPA32, anti-CK2, anti-GLP (Bethyl); anti-CK2, anti-RPA70, antiCpan-threonine, antiCpan-tyrosine, antiCp-Chk1(s354), anti-Chk1 (Cell Signaling Technology), and anti-53BP1 (Novus Biologicals). Generation of G9a Knockout Cell Lines. HCT116 cells were.