Fig. 6
RAF1 inhibitor sorafenib attenuates STOML2-induced CRC proliferation and tumor growth. a Immunoblot analysis of STOML2, PHB and MAPK signaling pathway in SW480-STOML2 cells, after administration of sorafenib (10 μM, 24 h) or DMSO. b Cell-cycle distribution analysis of STOML2-overexpressed SW480 and control after sorafenib (10 or 20 μM) or DMSO administration. Fraction of cells in G1/G2/S phase was shown. c CCK8 proliferation assay. SW480-STOML2 cells were seeded 5000 per well in 96-well culture plate; after administration of DMSO or sorafenib (10 or 20 μm), OD values were measured by day. Data are presented as means ± SEM from five independent experiments. ***P < 0.001, two-way ANOVA. d Colony formation of SW480-STOML2 cells. 5000/well of indicated cells were seeded in 6-well culture-plates. DMSO or 10, 20 μM sorafenib was administrated before Giemsa staining at 14th day culture. e Number of colonies formed on each plate. Data presented as means ± SEM from three independent experiments. ***P < 0.001, one-way ANOVA. f-n Subcutaneous implantation of MC38-STOML2 and MC38-mock cells in C57BL/6 mice with administration of sorafenib (10 or 20 μM) or DMSO. (f) Subcutaneous implantation of DMSO or 10, 20 μM sorafenib treated MC38-STOML2 cells in C57BL/6 mice. Tumor volume (cm3) measured at indicated time points. ***P < 0.001, two-way ANOVA. (g) 2 × 106 cells were injected into hind limbs of each group; tumors were retrieved at 21st day after injection. (h) Tumor weight (g) measured at retrieval. **P < 0.01, ***P < 0.001, t test. (i) Representative image of HE staining of subcutaneous implant in each group. Scale bar, 40 μm. (j) Representative images of IHC staining show negative correlation between Ki-67 and sorafenib dosage in each group. Scale bar, 40 μm. (k) Relative intensity of Ki-67 staining. **P < 0.01, ***P < 0.001, t test. (l) Subcutaneous implantation of DMSO or 10, 20 μM sorafenib treated MC38-STOML2 cells in C57BL/6 mice. Tumor volume (cm3) measured at indicated time points. **P < 0.01, ns, not significant, two-way ANOVA. (m) 2 × 106 cells were injected into hind limbs of each group; tumors were retrieved at 26th day after injection. (n) Tumor weight (g) measured at retrieval. ns, not significant, t test. o Schematic depiction of regulatory mechanism underlying CRC proliferation via STOML2/PHB/MAPK signaling and therapeutic target