Figure 3.

LAMC1-mediated preadipocytes differentiation promotes pre-metastatic niche formation and gastric cancer cell colonization in peritoneal microenvironment. (A) The Oil Red O staining and Western bolts for analyzing effect of different concentrations of LAMC1 (0, 50ng/ml and 100ng/ml) on 3T3-L1 and human omental preadipocytes differentiation for 5 days. (B) Cytokines secreted by 3T3-L1 after induction of different concentrations of LAMC1 for 24h were measured by RT-qPCR. (C) HSL expression in induced 3T3-L1 detected by RT-qPCR and western blots. (D) ELISA was used for analyzed FFAs, lipid droplets, adiponectin and leptin in 3T3-L1 CM after stimulation by different concentrations of LAMC1 for 4 days. (E) ELISA was used for analyzed FFAs in human omental preadipocytes CM after stimulation by different concentrations of LAMC1 for 4 days. (F) The flow chart of coculture. (G)ELISA for analyzing LAMC1 levels in supernatant of gastric cancer cell lines transfected with siLAMC1. (H) The lipid droplet formation ability of 3T3-L1 was measured by Oil Red O staining and adipocyte differentiation related protein after coculture for 5 days with gastric cancer cell supernatant transfected siLAMC1 for 48h. (I) Flowchart of reverse co-culture of 3T3-L1 supernatant and gastric cancer cells. (J) Western blots were used for E-cadherin and vimentin expression in gastric cells after coculture for 48h with 3T3-L1 supernatant induced by LAMC1 (0, 50ng/ml and 100ng/ml) for 4 days. (K) CCK8 assay for AGS cell proliferation detection. (L) The morphological characteristics of tumor xenograft model in AGS NC/si and 3T3-L1 coculture group, and tumor weight were shown. (M) The HE staining and immunohistochemical results of xenograft tumor in AGS NC/si and 3T3-L1 coculture group. Error bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001.