Fig. 4
Sema3C drives stemness maintenance and signaling via NRP1 and ITGB1. a Scheme displaying the procedure used for identifying the potential receptors that can bind to Sema3C and NRP1. b Co-IP analysis for validation of ITGB1 as a binding protein of Sema3C or NRP1 in Huh7 cells. c NRP1 and ITGB1 were knocked down in Huh7 cells respectively, and the interaction between Sema3C, NRP1 and ITGB1 was detected by Co-IP. d ITGB1 expression was detected in HCC cells when Sema3C was overexpressed or knocked down. e Correlation between ITGB1 expression and stemness-associated genes in HCC using the HCCDB datasets. The dots in the figure indicate p < 0.05. The color intensity and size of the circle are proportional to the correlation coefficients. f The expression levels of ITGB1 were determined by western blotting in ITGB1 knockdown Huh7 cells. Huh7 cells transfected with siITGB1. The chemoresistant effect was investigated by an MTT assay (g). The ability of self-renewal was determined by a sphere formation assay, scale bar, 100 μm (h). i Limiting dilution assay of Huh7 cells with or without ITGB1 knockdown (shITGB1) in BALB/c nude mice (n = 5 per group). Huh7 cells were transfected with OE-Sema3C or siITGB1. The chemoresistant effect was investigated by an MTT assay (j). The ability of self-renewal was determined by a sphere formation assay, scale bar, 100 μm (k). The protein expression levels of Gli1, p-AKT, AKT, and c-Myc were validated by western blotting (l). m Kaplan–Meier survival analysis for overall survival of HCC patients segregated according to high or low gene signatures for Sema3C + NRP1 + ITGB1. Co-IP Coimmunoprecipitation. OE overexpression. NC nontarget control. Data are presented as means ± SD. ns, not significantly *p < 0.05, **p < 0.01, ***p < 0.001
