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. 2021 Oct 28;78(23):7709–7732. doi: 10.1007/s00018-021-03997-2

Fig. 3.

Fig. 3

GRP78 is required for the M2 polarization of macrophages. A–F PBMC-derived macrophages were transfected with the indicated shRNAs by lentivirus. A The relative expression of GRP78 in each group of cells was measured by qPCR. N = 6. B The knockdown efficiency of GRP78 was further verified by western blotting, and β-actin was used as the loading control in western blotting. N = 6. C, D After shRNA transduction, PBMC-derived macrophages were polarized to M1 or M2 macrophages for 48 h. The relative expression of the M1 markers IL-1β, IL-12 and iNOS (C) and the M2 markers IL-10, Arg-1 and Mrc-1 (D) was determined by qPCR. N = 6. E, F shRNA-transfected cells were then cocultured with control medium, A549 CM, HCC827 CM or IL-4/IL-13 for another 48 h. E The polarization of M2 macrophages was analysed by FACS using the M2 surface marker CD11b+CD206+. N = 6. F Statistical analysis of the proportions of M2 macrophages in E. G–K Macrophages were untransfected or transfected with the indicated plasmid. G The expression of GRP78 was assessed by western blotting, and β-actin was used as the loading control. N = 6. The normalized GRP78 expression to β-actin is shown on the right. H–I After transfection, PBMC-derived macrophages were polarized to M1 or M2 macrophages for 48 h. The relative expression of the M2 markers IL-10, Arg-1, and Mrc-1 (H) and the M1 markers IL-1β, IL-12, and iNOS (I) was determined by qPCR. N = 6. J, K After transfection, PBMC-derived macrophages were then cultured without or with IL-4 and IL-13 for 48 h. J The polarization of M2 macrophages was analysed by FACS using the M2 surface marker CD11b+CD206+. N = 6. K Statistical analysis of the proportions of M2 macrophages in J. A, C, D, H, I mRNA expression was normalized to that of GAPDH, and the experiments were performed in triplicate. A–J The results are representative of three independent experiments. A, C, D, F, H, I, K Error bars represent the mean ± SD. p values were determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (A, C, D, H, I, K) or unpaired t test (F). ***p < 0.001, **p < 0.01, *p < 0.05