Fig 2: Decreased expression of RNF43 protein in GC stem-like cells. a Western blot assay of RNF43, RNF75, Cul4a, and Sox-2 protein in cancer cells and HGC-27 and N87 GC stem-like cells. b qPCR of RNF43 mRNA expression in cancer cells and CG stem-like cells. Results represent mean ± SD of three independent experiments. c Representative images of primary tumorsphere cells (upper, HSC034; bottom, HSC035). Most spheres were larger than 100 μm in diameter after 7 days of culture. P passage. d Western blot assay of RNF43 and Sox-2 protein in primary tumorsphere cells and corresponding cancer and adjacent normal tissues. e qPCR of RNF43 mRNA expression in primary tumorsphere cells and corresponding cancer and adjacent normal tissues. Results represent mean ± SD of three independent experiments. *means Statistically significant (P < 0.05); **means Statistically significant (P < 0.01)

Fig 3: SPC25 upregulation increases the incidence of DNA damage and activates the PI3KDNA-PK/AKT/Notch1 signaling pathway in HCC. (A-B) Images of γH2AX immunostaining in indicated HCC cells 72 hours after transfection with different plasmids. (C) Western blot analysis of the activation levels of DNA-PK, AKT, and Notch1 signaling pathway in indicated HCC cells. (D) Huh7-SPC25 and Hep3B-SPC25 cells were treated with 5 µM AZD-7648 and 5 µM MK-2206 respectively; changes in the activation of the DNA-PK/AKT and Notch1 signaling pathway and the expression levels of SOX2, NANOG, and CD133. (E) Huh7-SPC25 and Hep3B-SPC25 cells were transfected with Notch1-specific siRNAs, and changes in the activation of the DNA-PK/AKT and Notch1 signaling pathway and the expression levels of SOX2, NANOG, and CD133. (F-G) An RBP-Jκ binding site, as determined by the analysis of the SOX2 and NANOG promoters. CHIP was performed using IgG, NICD, and RBP-Jκ antibodies, followed by quantitative RT-PCR in indicated cells. Error bars represent the mean ± SD. *P < 0.05; **P < 0.01; *** P < 0.001; ****P < 0.0001; ns means non-significant.

Fig 4: RNF43 regulates the Wnt/β-catenin pathway in GCSLCs. a Western blot of RNF43 expression in cancer cell lines. RNF43 OE leads to downregulation of β-catenin, a key protein in Wnt/β-catenin pathway, as well as reduction of TCF4, C-myc, and Lgr5 expression. b Xenograft tumor tissues were analyzed for RNF43, β-catenin, and Sox-2 expression by immunohistochemistry. Scale bar, 10 μm (black) and 25 μm (red) (Color figure online). OE overexpression

Fig 5: TNFα increases breast cancer initiating cells and induces phenotype conversion in breast cancer cells. (A, B) Mammosphere formation assay was performed using SUM159PT mammospheres plated in a 96-well plate and treated with different concentrations of TNFα (10, 50, 100, 500 ng/mL) or vehicle (0.1% BSA). The number of mammospheres formed in each condition was counted and normalized against the vehicle control. Representative images of mammospheres in each condition (A) with the percentage of mammospheres formed (B) quantified. (C) The SUM159PT cells were plated at a density of 200 cells per well in a 6-well plate, then treated with a single dose of TNFα (1, 10, 50, 100 ng/mL) or vehicle (0.1% BSA) and cultured for 14 d. The resulting data were presented as plating efficiency with the percentage of colonies formed. (D) Sorted ZsGreen-cODC-negative SUM159PT ZsGreen-cODC vector expressing cells were plated at a density of 50,000 cells per well in a 6-well plate and the following day were pretreated with either with TNFα (1, 10, 50, 100 ng/mL) or vehicle (0.1% BSA) 1 h before irradiation at a single dose of 0, 4 or 8 Gy. Five days later, the cells were trypsinized and analyzed for ZsGreen-cODC-positive population by flow cytometry, with noninfected parental SUM159PT cells used as controls. (E) Sorted ZsGreen-cODC-negative SUM159PT cells were plated and pre-treated with 100 ng/mL TNFα or vehicle (0.1% BSA) 1 h before irradiation at a single dose of 0 or 4 or 8 Gy. The proteins were extracted 5 d later and were subjected to western blotting. The blots were analyzed for Oct4, Sox2, c-Myc, Klf4, and GAPDH, with GAPDH as the loading control. (F) The intensity of each band was quantified using ImageJ and presented as density ratio of gene over GAPDH. (G) Mammosphere formation assay were performed plating MCF-7 cells into 96-well plates, treated with different concentrations of TNFα (1, 2.5, 5, 10, 50, 100 ng/mL) or vehicle (0.1% BSA). The number of mammospheres formed in each condition was counted and quantified. (H) Sorted ZsGreen-cODC-negative MCF-7-ZsGreen-cODC cells were plated and the following day pretreated with either with TNFα (1, 2.5, 5, 10 ng/mL) or vehicle (0.1% BSA) 1 h before irradiation using a single dose of 0, 4 or 8 Gy. Five days later, the cells were trypsinized and analyzed for ZsGreen-cODC-positive cells by flow cytometry. (I, J) The MCF-7 cells were plated at a density of 200 cells per well into 6-well plates, treated with a single dose of TNFα (1, 2.5, 5, 10 ng/mL) or vehicle (0.1% BSA) and cultured for 14 d. Colony formation is shown in (I) and quantified and expressed as percentage of the number of cells pllated in (J). All experiments have been performed with at least 3 biological independent repeats. P values were calculated using 1-way ANOVA for B, C, G, and I; 2-way ANOVA for D, F, and H. *P value < 0.05, **P value < 0.01, ***P value < 0.001, and ****P value < 0.0001.