Fig. 4.

SOX2 overexpression reverses the VDR-mediated inhibition of stemness, and the vitamin D-VDR signaling pathway affects the stemness of CRC in acidic environments. a Immunoblotting of VDR and SOX2 in control and VDR-overexpressing CC tissue CSCs with or without SOX2 overexpression. b Tumor sphere formation assays (left) and limiting dilution assays (right) of control and VDR-overexpressing CC tissue CSCs with or without SOX2 overexpression. Student’s t-test. c Immunoblotting of SOX2, CD133, and CD44 in control and VDR-knockdown CC tissue-adherent cells treated with control or SOX2-targeting shRNA. d Tumor sphere formation assays of control and VDR-knockdown CC tissue-adherent cells treated with control or SOX2-targeting shRNA. Student’s t-test. e qPCR of VDR and SOX2 in control and VDR-knockdown CC tissue CSCs cultured under pH 7.4 and 6.8. Student’s t-test. f Tumor sphere formation assays of control and SOX2-knockdown CC tissue CSCs under pH 7.4 and 6.8. Student’s t-test. g Cell viability of control and SOX2-knockdown CC tissue CSCs with oxaliplatin treatment under pH 7.4 and 6.8. The IC50 is shown as a dotted line. h qPCR of SOX2, OCT4, MYC, CCND1, CD133, NANOG, CD44, and EPCAM in control and SOX2-knockdown CC tissue CSCs cultured under pH 7.4 and 6.8. Student’s t-test. i, j Representative images (i) and quantified data (j) for tumor spheres formed by CC tissue CSCs treated with 0, 50, or 100 nM of the active form of vitamin D (1α,25-(OH)-2-D3, VD₃) under pH 7.4 and 6.8. Scale bars: 200 μm. Student’s t-test. k Immunoblots (right) and quantified levels (left) of VDR, SOX2, and OCT4 in CC tissue CSCs treated with 0, 50, or 100 nM of the active form of VD₃ under pH 7.4 and 6.8. Student’s t-test. Three independent experiments were performed to obtain the data in b, d–h, j, and k. The data are shown as the mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001