Fig. 7
Verification of the pathway genes based on microarray analysis. A, A network regulation model of GCAFs promoting VM formation of GBC. NOX4, STAT3, JAK1 and JAK2 are regulated in the VM-related key genes. B, Confirmation of IL-6 expression in GCAFs/NFs in vitro (b1, Western blotting; b2, ELISA). IL-6 protein and product were highly secreted and upexpressed in GCAFs compared with NFs (all *P < 0.05). C-D, Verification the expression of IL-6/JAK/STAT3 pathway genes in vitro and in vivo. C (in vitro): c1 (ELISA), the expression of IL-6 product in the supernatant of GBC-SD + GCAFs co-culture was significantly higher than that of GBC-SD or GBC-SD + NFs co-culture (*P < 0.05); c2-c3 (c2, IHC, × 200; c3, western blotting), the expression of JAK1, JAK2 and STAT3 proteins in the 3-D co-culture matrices of GBC-SD + GCAFs was significantly higher than that of GBC-SD or GBC-SD + NFs co-culture (all *P < 0.05); c4 (qRT-PCR), the expression of JAK1, JAK2 and STAT3 mRNAs in the 3-D co-culture matrices of GBC-SD + GCAFs was significantly upregulated compared with GBC-SD or GBC-SD + NFs co-culture (all *P < 0.05). But, no statistical difference was observed in the expression of JAK1, JAK2 and STAT3 at protein or mRNA level between GBC-SD and GBC-SD + NFs groups. D (in vivo): d1 (ELISA), the expression of IL-6 product in the supernatant of GBC-SD + GCAFs group was significantly upregulated when compared with GBC-SD in xenografts (*P < 0.05); d2-d3 (d2, IHC, × 200; d3, western blotting), The expression of JAK1, JAK2 and STAT3 proteins in GBC-SD + GCAFs group was significantly higher than that of GBC-SD group in xenografts (*P < 0.05)