Fig. 7
p-FoxM1S25 translocates to the nucleus and activates genes for monocyte recruitment, TAM polarization, immune escape, and angiogenesis by direct activation. a, b A549S25E were treated with 1 μM trametinib, an inhibitor of MEK, for 48 h. a qRT-PCR was performed for interferon-stimulated genes (IFITM1, IF44L, and MX1), IL1A, IL1B, IL6, VEGFA, CXCL1, and FOXM1 in A549S25E cells. b Immunoblot analyses were performed using specific antibodies for FoxM1, p-Erk1/2, Erk1/2, c-Fos, c-Jun, IFITM1, IL1A, IL6, and β-actin. c, d A549S25E cells were treated with 15 μM ruxolitinib, a JAK inhibitor, for 48 h. c qRT-PCR was performed for IFITM1, IF44L, MX1, IL1A, IL1B, IL6, VEGFA, CXCL1, and FOXM1 in A549S25E cells. d Immunoblot analyses were performed using anti-FoxM1, anti-STAT1, anti-p-STAT1, anti-IFITM1, anti-IL1A, anti-IL6, and anti-β-actin. e Immunofluorescence was performed with A549 cells expressing WT, S25A, or S25E mutant of FoxM1. FoxM1 (green), RFP (red), and DNA (DAPI, blue) was displayed. Scale bar, 5 μm. f The quantification of the population of cells in the cytoplasm, nucleus, and both is presented on the left. The percentage of cells that exhibited positive RFP (red) staining was assessed with the following categories (N > C, RFP staining predominantly in the nucleus; N = C, similar RFF levels in both the nucleus and cytoplasm; N < C, RFP staining mainly in the cytoplasm). The population of RFP-positive cells specifically in the nucleus was plotted (right). n > 800. g-i ChIP assays for FoxM1 binding to the promoters of FOS (g, left), STAT1 (g, right), IL6 (h, left), VEGFA (h, right), and IFITM1 (i). Assays were performed on chromatin fragments using antibody to FoxM1 and normalized to pre-immune normal IgG. Immunoprecipitated fractions were assayed by qRT-PCR for binding the promoters of FOS, STAT1, IL6, VEGFA, and IFITM1. The qRT-PCR products were visualized in agarose-gel. j qRT-PCR was performed for STING, TBK1, and IRF3 in A549S25E cells. k Immunoblot analyses were performed using specific antibodies for STING, p-TBK1, TBK1, IRF3 and anti-β-actin. l ChIP assays were performed for FoxM1 binding to the promoters of STING in A549S25E cells. m ChIP assays for IRF3 binding to the promoters of IFITM1 in A549.S25E cells. Assays were performed on chromatin fragments using antibody to IRF3 and normalized to pre-immune normal IgG. Immunoprecipitated fractions were assayed by qRT-PCR for binding the promoters of IFITM1. Data are presented as mean ± SD of three independent experiments (significantly different from the experimental control). *p < 0.05; **p < 0.01; ***p < 0.001; (n = 3)