Fig. 6

STAT3 activation is crucial to the eCIRP-induced dysregulation of actin remodeling. RAW 264.7 cells were incubated with rmCIRP (1 µg/ml) for 24 h with or without stattic (3 µM). PBS was used as a control. A–C After treatment, the levels of p-STAT3 and ARP2 were evaluated by Western blotting. Data were obtained from two independent experiments and are expressed as the mean ± SD (n = 8–12/group). The PBS control was set to 1 for normalization. The groups were compared by one-way ANOVA and the SNK method. *p < 0.05 vs. PBS; ^#p < 0.05 vs. rmCIRP. D, E After treatment, phagocytosis was evaluated by adding pHrodo-labeled E. coli, and microscopy images were acquired at the end of the assay. Representative images are shown. Data were obtained from two independent experiments and are expressed as the mean ± SD (n = 9–11/group). The experiments were repeated five times. The PBS control was set to 100% for normalization. The groups were compared by one-way ANOVA and the SNK method. *p < 0.05 vs. PBS. ^#p < 0.05 vs. rmCIRP. F After treatment, the efficiency of phagocytosis was recorded, and the kinetics with 10-min intervals are presented. At the end of the assay, data were analyzed and are expressed as the mean ± SD (n = 4/group). The groups were compared by one-way ANOVA and the SNK method. *p < 0.05 vs. PBS. ^#p < 0.05 vs. rmCIRP. G After treatment, cells were fixed, and F-actin was stained with phalloidin. Representative confocal microscopy images are presented. Scale bar, 50 µm. H, I After treatment, the levels of p-cofilin were evaluated by Western blotting. Representative blots are shown. The experiments were repeated three times. Data were obtained from two independent experiments and are expressed as the mean ± SD (n = 6–8/group). The PBS control was set to 1 for normalization. The groups were compared by one-way ANOVA and the SNK method. *p < 0.05 vs. PBS control; ^#p < 0.05 vs. rmCIRP. J P-cofilin immunostaining. P-cofilin (red), F-actin (green), and the merged staining images are shown. Scale bar, 50 µm