Fig 2: Targeting USP1 promotes prostate cancer cells’ response to therapeutic agent enzalutamide. A, RV1 (left panel) or LNCAP (right panel) cells were treated with the indicated concentrations of ML323 for 48 hours and analyzed by western blotting for the indicated proteins. B, RV1 or LNCAP cells were treated with 5µmol/L ML323 for 48 h and mRNA levels of the indicated genes were detected (mean ± SEM of 3 independent experiments). *P < .05; **P < .01. C, RV1 cells were seeded at low density on the plate, and maintained for 2 wk. Numbers of colonies were counted in 5 high-power fields (mean ± SEM of 3 independent experiments). **P < .01. D, RV1 cells were stably expressed with or without Flag-KDM4A constructs and treated with either vehicle or USP1 inhibitor ML323 for 24 h. Cells were subjected to western blot to examine the indicated protein levels. E, Cells as described in (D) were treated with the indicated concentrations of enzalutamide and cell survival was determined (mean ± SD [n = 3]). F, RV1 cells stably expressing control (Ctrl) or USP1 shRNA with or without Flag-KDM4A were subjected to western blotting to detect the indicated protein levels. G, Cells as described in (F) were treated with the indicated concentrations of enzalutamide and cell survival was determined (mean ± SD [n = 3]). H, Schematic representation of how USP1 regulates androgen receptor (AR) and c-Myc activities through KDM4A

Fig 3: Knockdown of SNTB1 reduces stemness and tumorigenesis. (A) Enrichment plots from GSEA to examine the correlation between SNTB1 expression and Wnt/β-catenin signaling (left) or cancer stemness (right). The green curve denotes the ES curve. (B) TOP/FOP-flash luciferase activity assay assesses the Wnt/β-catenin signaling activity in SW620 and CW-2 cells with indicated shRNA transfection. (C) Evaluation of β-catenin expression in dissected tumor tissues from xenograft mice by immunostaining. Quantification of β-catenin expression in the nucleus is normalized against the total β-catenin level. (D) Western blot analysis is used to examine the β-catenin protein level in whole-cell lysate (total) and the nucleus fraction (nucleus). The quantification of β-catenin protein levels is normalized to the p84 (nucleus) and α-tubulin (total) levels, respectively. (E) Immunofluorescence assay examines the nuclear translocation of β-catenin upon treatment with the indicated shRNA. Cell nuclei are stained with DAPI dye (blue). β-catenin fluorescence intensity is analyzed and quantified by Image J (right panel) (200×). (F) Western blot assesses the expression of β-catenin downstream effectors c-Myc and CCND1 and cancer stem cell surface markers CD44 and CD24. Left, representative Western blot images; right, quantification of these protein levels expressed as fold change over the α-tubulin level. *, P<0.05. SNTB1, beta-1 syntrophin; GSEA, gene set enrichment analysis; ES, enrichment score; CCND1, cyclin D1.

Fig 4: p53 mutation enhances the resistance of CRC cells to PDT. p53 wt RKO and p53 mut SW480 cells were treated or non-treated with PDT and examined for cell viability by CCK-8 assay (A), cell apoptosis by Flow cytometry (B), and the expression levels of c-Myc (C), NEAT1 (D), miR-124 (E), and iASPP (F) by qRT-PCR. *P < 0.05, **P < 0.01, compared to RKO cells. ## P < 0.01 compared to RKO+PDT group.

Fig 5: C188 inhibits Wnt/β-catenin signaling by regulating the stability of β-catenin in breast cancer cells. (A and B) MCF7 and T47-D cells were treated with the indicated concentrations of C188 for 48 hours. DMSO was used as the control group. The phosphorylation and expression levels of Wnt pathway-related proteins were investigated by immunoblotting. β‑actin was used as a loading control. The results were representative of three independent experiment (**P<0.01). (C) MCF7 and T47-D cells were exposed to 40 μM C188 and 1 μM MG132 for 12 hours to evaluate whether MG132 could rescue the protein expression of β-catenin. β‑tubulin was loaded as a control (*P<0.05; **P<0.01). (D) Both MCF7 and T47-D cells were exposed to different concentrations of C188 for 48 hours, and then β-catenin localizing in the nucleus was detected with immunofluorescence. The nucleus was confirmed with DAPI (4,6-diamidino-2-phenylindole) staining. The red arrow represented the signal of β-catenin localizing in the nucleus. Scale bar: 10 μm. (E) MCF7 and T47-D cells were exposed to the indicated concentrations of C188 for 48 hours. DMSO was used as the control. The expression levels of Axin 2, c-Jun, and c-Myc proteins were evaluated by immunoblotting. β‑tubulin was used as a loading control. The results were representative of three independent experiment (**P<0.01; ***P<0.005).