Fig 2: PI3K/AKT signaling pathway mediated SETDB1-induced CSF-1 induction. a Western blotting of indicated proteins in U87 cells transfected with SETDB1 overexpression with or without MK-2206 pretreatment. b Western blotting of indicated proteins in U251 cells transfected with SETDB1 overexpression with or without MK-2206 pretreatment. c Western blotting of indicated proteins in U87 cells transfected with SETDB1 overexpression with or without siRNA against AKT. d Western blotting of indicated proteins in U251 cells transfected with SETDB1 overexpression with or without siRNA against AKT. e Western blotting of indicated proteins in U87 cells transfected with SETDB1 overexpression with or without rapamycin pretreatment. f Western blotting of indicated proteins in U251 cells transfected with SETDB1 overexpression with or without rapamycin pretreatment. g Western blotting of indicated proteins in control and SETDB1 overexpression xenograft tumors. h Western blotting of indicated proteins in control and SETDB1 knockdown xenograft tumors

Fig 3: SETDB1 regulates CSF-1 level in glioblastoma cells. a Real-time PCR for mRNA level of genes coding for tumor-associated macrophage (TAMs) recruitment associated cytokines in U87 cells with SETDB1 overexpression. b Real-time PCR for mRNA level of genes coding for tumor-associated macrophage (TAMs) recruitment associated cytokines in U251 cells with SETDB1 overexpression. c Real-time PCR for mRNA level of genes coding for tumor-associated macrophage (TAMs) recruitment associated cytokines in U87 cells with SETDB1 knockdown. d Real-time PCR for mRNA level of genes coding for tumor-associated macrophage (TAMs) recruitment associated cytokines in U251 cells with SETDB1 knockdown. e Enzyme-linked immunosorbent assay (ELISA) analysis of CSF-1 concentration in the supernatants of cultured glioblastoma cells with SETDB1 overexpression. f Enzyme-linked immunosorbent assay (ELISA) analysis of CSF-1 concentration in the supernatants of cultured glioblastoma cells with SETDB1 knockdown. g TCGA database indicated the correlation of CD163 and CSF-1 in glioblastoma tissues. h TCGA database indicated the correlation of SETDB1 and CSF-1 in glioblastoma tissues. Results were expressed as means ± SD of 3 independent experiments. **, P < 0.01

Fig 4: SETDB1 promotes cell growth, and apoptosis in vitro and in vivo. a mRNA level of SETDB1 in indicated cells transfected with SETDB1 plasmid or control vector. b Western blotting of SETDB1 in indicated cells transfected with SETDB1 plasmid or control vector. c CCK-8 of indicated cells transfected with SETDB1 plasmid or control vector. d Colony formation of indicated cells transfected with SETDB1 plasmid or control vector. e Western blotting of indicated proteins in U251 cells transfected with SETDB1 plasmid or control vector treated with 500 nM staurosporine (STS) for 24 h. f Ectopic expression of SETDB1 accelerated growth of U251 xenografts in nude mice (n = 6) as compared to controls. g Western blotting of ectopic expression of SETDB1 in tumors from U251-SETDB1 groups. h Representative images of Ki67-positive cells in vector and SETDB1 transfected tumors. Scale bar: 50 µm. Results were expressed as means ± SD of 3 independent experiments. **, P < 0.01

Fig 5: SETDB1 promotes macrophage recruitment and polarization. a Transwell migration assay of macrophage by CM from indicated U87 cells. b Real-time PCR for the expression levels of CD68 and CD163 in macrophages treated with CM from U87 cells as indicated. c Flow cytometry analysis for the expression levels of CD163 in macrophages treated with CM from glioblastoma cells as indicated. d Real-time PCR for the mRNA expression of tumor-associated macrophage (TAM) characteristic cytokines in macrophages treated with CM from glioblastoma cells as indicated. e Enzyme-linked immunosorbent assay (ELISA) for the secretion of tumor-associated macrophage (TAM) characteristic cytokines in macrophages treated with CM from U87 cells as indicated. f Real-time PCR for the mRNA expression of M1-related cytokines in macrophages treated with CM from U87 cells as indicated. g Enzyme-linked immunosorbent assay (ELISA) for the secretion of M1-related cytokines in macrophages treated with CM from U87 cells as indicated. Results were expressed as means ± SD of 3 independent experiments. **, P < 0.01