Fig. 4

G9a dimethylation of H3K9 requires endogenous cyclin D1. a, b Confocal microscopy of immunofluorescence for H3K9me2 (red) and nuclear staining with DAPI (blue) in G9a^fl/fl and G9a^−/− mouse embryonic fibroblasts (MEFs), and G9a^−/− MEFs rescued with G9a^WT or vector control. Images show the reduction in H3K9me2 in G9a^−/− cells (a) and quantitative analysis was shown as mean ± SEM (b). c Western blot for H3K9me2 and G9a in G9a^fl/fl and G9a^−/− MEFs. The G9a^fl/fl, G9a^−/−, and G9a^−/− MEFs rescued with G9a and vector control were assessed by western blot for H3K9me2. Lamin B1 was used as a protein loading control. S.E. shorter exposure, L.E. longer exposure. d Quantitation of H3K9me2 is shown as mean ± SEM for N = 3. e MCF-7 cells transduced with two individual shG9a and shGFP control were assessed by western blot for H3K9me2, cyclin D1, and G9a. Lamin B1 was used as a protein loading control. f, g Confocal microscopy of immunofluorescence for H3K9me2 (green) and nuclear staining with DAPI (blue) in G9a^−/− MEFs rescued with G9a^WT or vector control treated with cyclin D1 small interfering RNA. Images show the reduction in H3K9me2 by cyclin D1 siRNA in G9a^−/− plus G9a cells but not in G9a^−/− plus vector cells. Scale bar, 20 μm (f) and quantitative analysis was shown as mean ± SEM (g)