Figure 5.

HSP47-mediated COL1 biosynthesis promotes M2 microglial polarization through β-integrin/NF-κB signaling

(A) BV2 cells were treated with COL1 and then subjected to RNA sequencing analysis (n = 3 biological replicates for each group). The Kyoto Encyclopedia of Genes and Genomes analysis shows the top 11 enriched pathways with significant difference.

(B) Gene set enrichment analysis of the changed genes after COL1 treatment. NES (normalized enrichment score) and p value are shown.

(C) Heatmap showing the most significantly upregulated cytokine genes in COL1-treated BV2 cells vs. control BV2 cells.

(D) BV2 cells transfected with p65 small interfering RNA (siRNA) were treated with COL1, and the cell lysates were analyzed by immunoblotting. The levels of selected cytokine genes were analyzed by RT-qPCR.

(E) BV2 cells under COL1 treatment were treated with TC-I-15. Cell lysates were analyzed by immunoblotting, and the levels of selected cytokine genes were detected by RT-qPCR.

(D and E) GAPDH was used as an internal control. Data are expressed as mean ± SEM of n = 3 independent experiments. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

(F) CUT&Tag analysis of p65 binding at CCL2, CXCL2, and CXCL3 promoters in BV2 cells after treatment by COL1. Spike-in was used as an internal control. Data are expressed as mean ± SEM, n = 3 independent experiments, two-tailed Student’s t test. ∗∗∗p < 0.001.

(G) BV2 cells under COL1 treatment were transfected with p65 siRNA or treated with TC-I-15 and the expression of CD206 was analyzed by FACS. Representative FACS plots are shown. The FACS data were statistically analyzed (mean ± SEM, n = 3 independent experiments). ∗∗∗p < 0.001.