Fig. 6
IL-6 and cholesterol biosynthesis are responsible for Sema3C-mediated HSCs activation. a The volcano map showed the differentially expressed genes of LX-2 cells treated with rhSema3C or PBS in vitro. HSCs were treated with rhSema3C or supernatants of MHCC-97L cells with different Sema3C expression levels. mRNA levels of IL6 and IL8 were detected using qRT-PCR (b). ELISA was used to examine the IL6 secretion level (c). d GSEA identified an enrichment of genes involved in the NF-κB pathway in rhSema3C-treated LX-2 cells. e Western blotting showed phosphorylated and total p65 protein expression levels in LX-2 cells treated with rhSema3C. LX-2 cells were pre-treated with Bay 11-7082 and subsequently stimulated with rhSema3C, the levels of phosphorylated p65, total p65, and α-SMA expression were determined by western blotting (f), and IL6 secretion levels were examined by ELISA assay (g). h LX-2 cells were treated with OE-Sema3C-Hep3B cell-derived supernatants, Co-IP assay was used to detect the interaction between ITGB1 and NRP1. LX-2 cells were transfected with siNRP1 and subsequently stimulated with rhSema3C, IL6 secretion levels were detected by ELISA (i). The phosphorylated p65, total p65, α-SMA, and HMGCR expression levels were determined by using western blotting (j). k LX-2 cells were treated with a dosage of rhSema3C or supernatants of Sema3C-overexpressed HCC cells, and ITGB1 protein expression was determined by western blotting. LX-2 cells were transfected with siITGB1 and subsequently stimulated with rhSema3C, and the IL6 secretion levels were detected by ELISA assay (l), the phosphorylated p65, total p65, and α-SMA expression levels were determined by using Western blotting (m). LX-2 cells were treated with siNRP1, siITGB1 or rhSema3C as indicated, the secretion of TGF-β1 in each group was detected by ELISA (n), the chemotherapy resistance of LX-2 cells was evaluated by MTT assay (o), and migration and invasion were performed by Transwell assay, scale bar, 200 μm (p). rhSema3C recombinant human Sema3C; Data are presented as means ± SD. ns not significantly; *P < 0.05, **P < 0.01, and ***P < 0.001
