Figure 4: Chronically active G9a forms a stable complex with IFI35, NMI, and c-Myc during ET.

(a) IFI35 remained active in LPS-tolerant cells. mRNA expression was normalized to GAPDH. The release of TNFα indicates the strength of immune response. **P<0.01. Two-tailed unpaired Student’s t-test. (b) and c-Myc (d) specifically interact with G9a in LPS-tolerant, TLR4/CD14/MD2 293 cells. (c) IFI35 interacts with the catalytic SET domain of G9a in TL cells as assayed by IP with HA-tagged SET (upper), and vice versa (lower). (e) NMI, IFI35, c-Myc and G9a were co-immunoprecipitated from LPS-tolerant cells. (f) Both IFI35 and c-Myc associate with the peptide containing H3K9me2 in the complex with G9a. (g) c-Myc promotes the HMTase activity of G9a. c-Myc and G9a were co-transfected into LPS-tolerant, TLR4/CD14/MD2 293 cells and c-Myc was enriched for the H3K9 HMTase activity assay. ‘NTC’ refers to non-transfected control cells, EV is the EV control, HA-G9a represents the IP background and FLAG-G9a is a positive control for HMTase activity. Recombinant G9a (rG9a) is a positive control for in vitro reactions, and the reactions either without enzyme (W/O E) or without S-adenosyl methionine (W/O SAM) are negative controls. (h) G9a significantly repressed NF-κB reporter genes in a dose-dependent manner in cooperation with c-Myc. All luciferase assays were performed with three individual preparations. Values and error bars indicate the mean and s.d. among biological triplicates. The ectopic proteins were detected by immunoblotting. β-Actin is the loading control.