Tumor-associated macrophages-educated reparative macrophages promote diabetic wound healing (2022)
Figure 3

Figure 3 - TAMEMs resolve inflammation and stimulate angiogenesis and fibroblast growth in vitro.

A Quantification of concentrations of 22 soluble proteins from the cytokine and angiogenesis array analysis (*P < 0.05, ***P < 0.001, and ****P < 0.0001; ns: not significan; n=3, biological replicates).

B, C Flow cytometry analysis (B) and IF staining (C) for CD86 and CD206 in BMDMs that were cultured with conditional medium (CM) from TAMEMs, M2, M1, M0, and normal medium for 48 h (In B, ****P < 0.0001; In C, large and small images are derived from the same cell, the former show the CD86/CD206 channel, while the latter show the merger of F4/80 and CD86/CD206, scale bar: 20 μm; n=6, biological replicates).

D The real-time qPCR analysis of macrophage polarization genes (M1: Nos2 and Il6; M2: Arg1 and Mrc1) in BMDMs treated with CMs from TAMEMs and other controls for 48 h (*P < 0.05, ***P < 0.001, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the normal medium group; n=6, biological replicates).

E The real-time qPCR analysis of pro-inflammatory cytokines (Il6 and Cxcl15) in fibroblasts (L929) that pre-treated with LPS and then cultured with CMs from TAMEMs and other controls for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the LPS-treated group; n=6, biological replicates).

F, G Western Blotting analysis of phosphorylated IκB (F) and quantification of gray value (G) in fibroblasts (L929) that pre-treated with LPS and then cultured with CMs from TAMEMs and other controls for 24 h (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the LPS-treated group; n=3, biological replicates).

H Representative images and quantification of the Matrigel-based angiogenesis assay in mouse vascular endothelial cell (SVEC4-10) treated with CMs from TAMEMs and other controls, VEGF treatment as a positive control (****P < 0.0001 and ns: not significant (P > 0.05) vs. the normal medium group; scale bar: 100 μm; n=6, biological replicates).

I Scratch wound assay of L929 cells treated with different CMs for 4 and 8 h (scale bar: 200 μm; n=6). J Quantification of the remaining scratch area was performed using an image analysis, the rates are presented as a percentage of the initial scratch area at 0 h (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the normal medium group; n=6, biological replicates).

K The proliferation rate of L929 cells pretreated with different CMs was analyzed using the CCK-8 kit (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the normal medium group; n=6, biological replicates).

L-O The real-time qPCR analysis (L), Western Blotting (M) (including quantification of gray value (N)), and IF staining (O) for α-SMA and type I Collagen in L929 cells after 48 h treatment with different CMs (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the normal medium group, scale bar: 10 μm; n=3, biological replicates).

Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (D, H) and two-way ANOVA with Tukey's multiple comparison test in (A, B, E, G, J, K, L, N) in Graph Pad Prism 8.