Supplementary Figure S10 from Blocking M2-Like Macrophage Polarization Using Decoy Oligodeoxynucleotide-Based Gene Therapy Prevents Immune Evasion for Pancreatic Cancer Treatment

Supplementary Figure S10. JAK/STAT6/HDAC2 is the downstream signaling in regulating CYBB expression in THP-1 cells. A, RT-qPCR analysis for IL-4 expression was performed in THP-1 cells co-cultured with different pancreatic cell lines. The bar graphs depict relative mRNA expression. ***, p < 0.001, significant differences between groups. Data represent the mean of 3 independent experiments. B, RT-qPCR analysis for CD86 and MSR1(CD204) expression was performed in THP-1 cells co-cultured with different pancreatic cell lines. The bar graphs depict relative mRNA expression. ***, p < 0.001, significant differences between groups. Data represent the mean of 3 independent experiments. C, THP-1 cells were cocultured with PANC-1 cells and simultaneously treated with IL-4 neutralizing antibodies. After 72 h of incubation, THP-1 cells were collected for Western blotting to measure M1/M2 markers, CYBB, and GAPDH protein expression. D, Time-dependent effect of IL-4 (20 ng/mL) on CD86, CD204, CD206 (MRC1), STAT6, HDAC2, and CYBB protein expression was determined by Western blotting. E, THP-1 cells were pre-treated with the JAK1 inhibitor (Ruxolitinib, 10 μM), the JAK3 inhibitor (Tofacitinib, 50 μM), the STAT6 inhibitor (AS1517499, 250 nM), and the HDAC inhibitor (TSA, 10 uM) for 24h and then were treated with IL-4 (20 ng/mL) for 1h. Cell lysates were collected for Western blot analysis to measure indicated protein expression.