Fig 3 -

Mtb induced G9a-SREBP2 and SIRT6 transcriptionally regulate cholesterol biosynthesis and transport genes (A) SREBP2 was immunoprecipitated in whole cell lysates of BALB/c mouse peritoneal macrophages infected with H37Rv for 12 h to assess its interaction with G9a by immunoblotting. (B) Sequential ChIP was performed to assess the co-recruitment of SREBP2 and G9a at the promoters of Lrp2, Aacs, Hmgcs1, Mvd and Dhcr24 in mouse peritoneal macrophages infected with H37Rv for 12 h. (C) ChIP assay was performed to affirm the enrichment of H3K9me1 mark, on the promoters of the indicated genes upon 12 h infection with H37Rv in BALB/c mouse peritoneal macrophages. (D, E) BALB/c mouse peritoneal macrophages were transfected with Srebpf2 siRNA and (D) infected for 12 h with H37Rv to assess the expression of cholesterol accumulation genes by qRT-PCR, or (E) infected with H37Rv for 48 h (MOI 1:5) to analyze the in vitro CFU. (F-G) BALB/c mouse peritoneal macrophages infected with H37Rv for 12 h were analyzed by ChIP for (F) SIRT6 recruitment and (G) H3K9Ac mark, on the promoters of Abca1 and Abcg1. (H) BALB/c mouse peritoneal macrophages were infected with H37Rv for 12 h, and assessed for the recruitment of SIRT6, G9a and presence of H3K9Ac, H3K9me2 and H3K9me1, on the promoters of Abca1 and Abcg1. (I) BALB/c mouse peritoneal macrophages were infected with H37Rv for 24h and assessed for recruitment of SIRT6, G9a, H3K9Ac and H3K9me2 on promoters of Abca1 and Abcg1. (J) BALB/c mouse peritoneal macrophages were infected with H37Rv for 48h and assessed for recruitment of G9a and H3K9me2 on promoters of Abca1 and Abcg1.The MOI of infection is 1:10 (macrophage: mycobacteria) for all the in vitro experiments. All data represent the mean ± SEM from 3 independent experiments. The blots are representative of 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant (One-way ANOVA for B, D, Student’s t-test for C, E-J). Med, Medium; NT, non-targeting.