Fig 2: G9a expression in cervical cancer cells and the anti-cell proliferation effect of G9a chemical inhibitor BIX01294(A) Nuclear protein (10 μg) from the exponential phase of cervical cancer or normal human cervical epithelial cells (N1 and N2; 2 different lots) were used for G9a determination by western blot. Histone H3 was used as a loading control. L: long isoform. S: short isoform. (B) Effect of G9a chemical inhibitor BIX01294 on the methylation of histone 3 lysine 9. Histone H3 was used as a loading control. v: vehicle. The BIX01294 concentrations were 0 (vehicle), 1, 2.5, and 5 μM, respectively. (C) Effect of BIX01294 on the cell growth of cervical cancer cell lines. Cells were treated with 5 μM of BIX01294 at different times: viable cells were determined by MTT assay. Relative cell growth rates from day 0 were calculated. n = 5, *p < 0.05; **p < 0.01;****p < 0.0001. Data are presented as mean ± SD. (D) Effect of BIX01294 on the cell cycle of cervical cancer cell lines. Cells were treated with 5 μM of BIX01294: after 3 days, cell cycle was determined by propidium iodide staining. Different cell cycle phases are quantified. n = 5, ****p < 0.0001. Data are presented as mean ± SD.

Fig 3: Knockdown of either G9a or GLP does not have a significant effect on basal or TNFα-induced glycerol release in 3T3-L1 adipocytes. Knockdown of G9a or GLP, using G9a or GLP siRNA, and cell treatments were performed as described in the Figure 1 legend. Glycerol content was measured using 25 μL of conditioned medium. Statistical significance was determined using an unpaired Student’s t-test. This is a representative figure of an experiment independently performed three times on different groups of adipocytes.

Fig 4: G9a and cyclin D1 bind common regulatory regions of genes in chromatin immunoprecipitation (ChIP)-Seq in MCF-7 cells. a, b ChIP-qPCR analysis of target genes Pttg1 and c-Myc in MCF-7 cells for G9a and cyclin D1 binding. c Western blot for cyclin D1 in cyclin D1 siRNA-treated MCF-7 cells confirmed decrease of cyclin D1 abundance. d–f Abundance of mRNA for Mdm4 (d), Pttg1 (e), and c-Myc (f) in MCF-7 with cyclin D1 siRNA or control is shown. Data of qPCR is mean ± SEM for n = 3. Eth ethanol, E2 estradiol. g–i ChIP-qPCR analysis of target genes Pttg1 and c-Myc for G9a binding in cyclin D1 rescue cyclin D1−/− mouse embryonic fibroblasts

Fig 5: G9a and cyclin D1 bind common regulatory regions of genes in chromatin immunoprecipitation (ChIP)-Seq. a Venn diagram depicting the overlapping intervals shared between cyclin D1 ChIP-Seq and G9a ChIP-Seq. b Gene Ontology (GO) biological function enrichment scores for overlapping terms for 744 genes common between cyclin D1 ChIP-Seq and G9a ChIP-Seq. c–k Three genes (Mdm4, Pttg1, and Myc) were selected from the “oncogene” GO biological term and eight genes were selected from the “neuronal activities” GO term (Supplemental Figs. 4,5). c, f, i Depicted are tag density profiles for cyclin D1 intervals (red) and G9a intervals (blue) with respect to the identified genes. Profiles generated by Integrated Genome Browser are depicted for enriched regions binding G9a and the same region of cyclin D1 ChIP-Seq. Enriched intervals are designated by an * for cyclin D1 and a * for G9a. Tag density profiles are not drawn to scale (c, f, i). d, g, j Individual ChIP-qPCR analysis of target genes identified in ChIP-Seq. FLAG (FLAG-cyclin D1) ChIP-qPCR analysis of target genes in cyclin D1−/− plus GFP vector vs cyclin D1−/− plus cyclin D1WT rescue mouse embryonic fibroblasts (MEFs), and e, h, k G9a ChIP-qPCR of the same target genes in G9a−/− plus vector vs G9a−/− plus G9aWT rescued MEFs. H3K9me2 ChIP-qPCR is conducted in each cell type with IgG as control. Data are shown as mean ± SEM for ChIP-qPCR of FLAG (FLAG-cyclin D1) and H3K9me2 for target genes identified in ChIP-Seq. Significant difference are shown as **P < 0.01 or *P < 0.05 for cyclin D1−/− plus GFP vector vs cyclin D1−/− plus cyclin D1WT (d, g, j). Mean ± SEM is shown for ChIP-qPCR of G9a and H3K9me2 for Mm44, Pttg1, and c-Myc genes. Significant difference is shown as **P < 0.01 or *P < 0.05 for G9a−/− plus vector vs G9a−/− plus G9aWT (e, h, k)