Fig 1: Effects of xCT knockdown on the stemness of HCT116 and HCT15 CRC cells.A An overview of the correlations between xCT expression, mRNAsi, and known clinical characteristics (TNM stage, sex, and survival status) in the TCGA-COAD cohort. B The association between the expression of xCT and mRNAsi in the TCGA-COAD cohort. C, D Western blot assay was applied to detect the expression of xCT in CRC cells and CRC stem cells. E, F Sphere formation assays showed that xCT inhibition suppressed the sphere-forming capability of CRC stem cells. G, H Western blot analysis showed that xCT knockdown reduced the expression level of stemness-related molecules in HCT116 cells and HCT15 cells. I, J Flow cytometry indicated that xCT inhibition significantly diminished the expression of EPCAM and CD133 in CRC cells. K, L IF staining showed that xCT knockdown suppressed the expression of CD133 in CRC cells. **P < 0.01.
Fig 2: SchB alleviates the stemness of cancer stem-like cells. Cancer stem-like cells (NCI-H460-CSCs and H661-CSCs) were treated with 10, 20 or 40 µmol/l SchB and the control group was treated with PBS. (A) Flow cytometry was used to evaluate the number of CD133+ cells. (B) CD133, CD44, Oct-4 and Bmi-1 expression was tested by western blot analysis. (C) Sphere-forming ability of cancer stem-like cells was estimated by sphere-forming assay. Scale bar, 25 µm. All experimental data are shown as the mean ± SD (n=3). Differences were analyzed by one-way ANOVA with Tukey's correction. *P<0.05, **P<0.01 vs. control. SSC, side scatter; SchB, Schisandrin B; Oct-4, octamer-binding transcription factor 4; Bmi-1, B lymphoma Mo-MLV insertion region 1 homolog.
Fig 3: Silencing of xCT suppresses CRC growth and blocks spontaneous lung metastasis of CRC cells.A xCT knockdown inhibited the growth of tumors in CRC xenograft mouse models. Differences in tumor weight (B) and volume (C) in nude mouse CRC models subcutaneously injected with HCT116 cells with or without xCT inhibition. D IHC staining showed the changes in the expression of Ki67, N-cadherin, E-cadherin, CD133 and P-AKT in CRC tissues after xCT knockdown. E The process to establish a CRC lung metastasis mouse model. F qRT-PCR and western blotting assays confirmed the knockdown efficiency with xCT shRNA administration in CT26 cells. Macroscopic changes in the lungs (G) and pathological changes in the lung tissues (H) of mice after caudal vein injection of CT26 cells with or without xCT inhibition. I xCT knockdown significantly inhibited the metastatic capability of CRC. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: RAI2 inhibited CRC stem cell-like properties and increased the chemosensitivity of CRC cells to 5-FU and L-OHP. (A) Left panel: Representative images showing CD133-positive cell proportion before and after fluorescence-activating cell sorting by CD133 antibody in LoVo/HCT116 cells. Right panel: Representative images showing sphere-forming ability affected by RAI2 re-expression in LoVo/HCT116 cells. (B) Left panel: Representative images showing CD133-positive cell proportion before and after fluorescence-activating cell sorting by CD133 antibody in LoVo/HCT116 cells. Right panel: Representative images showing sphere-forming ability affected by RAI2-M re-expression in LoVo/HCT116 cells. (C) Western blot of CD133, SOX2 and OCT4 expression in RAI2 re-expressed or vector control cells. (D) Western blot of CD133, SOX2 and OCT4 expression in RAI2-M re-expressed or vector control cells. (E) Representative curves of growth inhibitory effects of 5-FU in LoVo/HCT116 cells with or without re-expression of RAI2/RAI2-M in different concentrations (0.00, 6.25, 12.50, 25.00, 50.00, 100.00μM). (F) Representative curves of growth inhibitory effects of L-OHP in LoVo/HCT116 cells with or without re-expression of RAI2/RAI2M in different concentrations (0.00, 1.00, 2.00, 4.00, 8.00, 16.00μM). (G) Growth inhibitory effect of 5-FU/L-OHP in SW620 cells with or without RAI2 knocked down and SW620 cells with both shRAI2 and RAI2-M transfected. The treatment concentrations of 5-FU in SW620 cells were (0.00, 6.25, 12.50, 25.00, 50.00, 100.00μM), and the treatment concentrations of L-OHP were (0.00, 2.00, 4.00, 8.00, 16.00, 32.00μM). The viability of cells was measured by MTT assay after 5-FU/L-OHP treatment for 48 hrs. p-values: *≤0.05; **<0.01.
Fig 5: SchB leads to toxicity in cancer stem-like cells. To induce cancer stem-like cells, NCI-H460 and H661 cells were incubated in serum-free DMEM/F12 medium containing appropriate growth factors. (A) Following induction, the ratio of CD133+ cells was analyzed by flow cytometry. Blue cells represent CD133− cells and red cells represent CD133+ cells. (B) Expression levels of CSC markers (CD44, Oct-4 and Bmi-1) were detected by western blot analysis. (C) Sphere-forming assay was performed to estimate CSC traits of large-cell lung cancer cells. Scale bar, 25 µm. **P<0.01 vs. parental cells. (D) Cell cycle was evaluated by flow cytometry. **P<0.01 G0/G1 parental cells vs. cancer stem-like cells. (E) Chemical structure of SchB. (F) Cancer stem-like cells and parental cells were incubated with different doses of SchB (0, 5, 10, 20, 40, 80, 120 and 160 µmol/l) for 72 h and Cell Counting Kit-8 assay was utilized to examine the viability of cancer stem-like cells and parental cells. Cells treated with 0 µmol/l SchB acted as control. Differences were analyzed via one-way ANOVA with Tukey's correction. *P<0.05 and **P<0.01 vs. 0 µmol/l. All experimental data are shown as the mean ± SD (n=3). SSC, side scatter; SchB, Schisandrin B; CSC, cancer stem cell; Oct-4, octamer-binding transcription factor 4; Bmi-1, B lymphoma Mo-MLV insertion region 1 homolog.
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