Fig. 4: Fra-1 regulation of HMGA2 expression promotes CCL2 binding to CCR2 and induces macrophage M2-type polarization.

A, B HMGA2 was overexpressed in gastric cancer cells, and the effect of HMGA2 on CCL2 secretion was detected using an ELISA assay kit. The p-value was calculated through unpaired t-test correction. C, D In gastric cancer cells, Fra-1 was overexpressed, and Fra-1 was also overexpressed while HMGA2 was knocked down. The effect of Fra-1-regulated HMGA2 expression on CCL2 secretion was detected using an ELISA assay kit. Multiple comparisons were analyzed using ANOVA, and two groups were compared by Wilcoxon rank sum test. E, F In gastric cancer cells HGC27, HMGA2 overexpression was performed, and the effect of HMGA2 on CCL2 mRNA levels was detected using RT-qPCR. The p-value was calculated through unpaired t-test correction. G, H In gastric cancer cells HGC27, HMGA2 overexpression was performed, and the effect of HMGA2 on CCL2 protein expression levels was detected using Western blot. I, J In gastric cancer cells AGS, HMGA2 overexpression was performed, and the effect of HMGA2 on CCL2 mRNA levels was detected using RT-qPCR. Multiple comparisons were analyzed using ANOVA, and two groups were compared by Wilcoxon rank sum test. K, L In gastric cancer cells AGS, HMGA2 overexpression was performed, and the effect of HMGA2 on CCL2 protein expression levels was detected using Western blot. M M0 macrophages were co-cultured with conditioned medium from gastric cancer cells overexpressing Fra-1, and the co-localization of CCL2 secreted by gastric cancer cells and CCR2 expressed by macrophages was detected using laser confocal microscopy. N M0 macrophages were induced to polarize to the M2 type by adding IL4 (20 ng/mL) and IL13 (20 ng/mL) to the medium, and the induction efficiency was detected using flow cytometry. The p-value was calculated through unpaired t-test correction. O Successfully induced M2-type macrophages were co-cultured with conditioned medium from gastric cancer cells overexpressing Fra-1, with (or without) the addition of the CCR2 antagonist INCB3344 (5 nM), and the co-localization of CCL2 secreted by gastric cancer cells and CCR2 expressed by macrophages was detected using laser confocal microscopy. P, Q Successfully induced M2-type macrophages were co-cultured with conditioned medium from gastric cancer cells overexpressing Fra-1, with (or without) the CCR2 antagonist INCB3344, and the percentage of M2-type macrophages polarized was detected using flow cytometry. Multiple comparisons were analyzed using ANOVA, and two groups were compared by Wilcoxon rank sum test. R–U Successfully induced M2-type macrophages were co-cultured with conditioned medium from gastric cancer cells overexpressing Fra-1, with (or without) the addition of the CCR2 antagonist INCB3344, and the secretion of the M2-type macrophage marker cytokines TGF-β, Arg-1, and IL-10 was detected using an ELISA assay kit. Multiple comparisons were analyzed using ANOVA, and two groups were compared by Wilcoxon rank sum test. All experiments were performed with three or more independent replications, and the data shown are from representative individual experiments. “ns” indicates not significantly different; “*“ indicates p < 0.05; “**“ indicates p < 0.01; “***“ indicates p < 0.001; “****“ indicates p < 0.0001.