Fig. 3: M1-induced apoptosis via the IFN-β signaling pathway.

A Cytokine profiling of M0 and M1 CM. High-glucose RPMI supplied with 10% FBS is served as the medium control. B Effect of IFN-β, IFN-γ or combination treatments on p53 expression in A549 cells. Cells were treated with 1 ng/ml IFN-γ and/or 5, 10, and 20 ng/ml IFN-β for 3 days. The lower panel shows p53 protein levels normalized to β-actin, quantified by ImageJ. C IFN-β levels in M0 and M1 CM measured by ELISA. High-glucose RPMI supplied with 10% FBS is served as the medium control. D Synergistic effect of IFN-β and IFN-γ on apoptosis in A549 cells. Cells were treated with IFNs at indicated concentrations for 3 days. E Inhibition of M1-induced apoptosis through IFN-β neutralization. The combination treatment included 2 μg/ml each of IFN-β and IFN-γ neutralizing antibodies (nAb) and 4 μg/ml IgG as the negative control. F Effect of JAK inhibitor I on M1-induced apoptosis in A549 cells. DMSO served as the negative control. G Effect of JAK1, JAK2, and TYK2 silencing on M1-induced apoptosis in A549 cells. NC: siRNA negative control. H Effect of IFNAR1 and IFNAR2 silencing on M1-induced apoptosis in A549 cells. NC: siRNA negative control. I Inhibition of M1-induced apoptosis through IFNAR1 and IFNAR2 neutralization. M1 CM was supplied with neutralizing antibodies or 2 μg/ml IgG. Apoptotic cells were quantified using Annexin V staining and assessed by flow cytometry. Data are representative of at least two independent experiments and represented as mean ± SD. P-value for multiple group comparisons determined by two-way ANOVA, otherwise using one-way ANOVA and followed by Tukey’s post hoc test. ns not significant, P-value > 0.05; *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001. See Supplementary Fig. 2A for gating strategy of apoptosis assay.