Fig 2: Haploid knockout of PIK3R1 activates WNT/ß-catenin pathway dependent on the phosphorylation of AKT.(a), The RT-PCR analysis of the mRNA expression of PIK3R1, CTNNB1, HES1, GLI1, and NANOG in 786-O, 786-mut1, 786-mut2, A-704, A-704-mut1, and A-704-mut2 cells. (b), 786-O, 786-mut1, 786-mut2, A-704, A-704-mut1, and A-704-mut2 cell lysates were immunoprecipitated by anti-PIK3R1 antibody and immunoblotted with anti-PIK3R1 (upper panel) and anti-P110 antibody (middle panel). Whole lysates immunobotted with anti-P110 antibody (lower panel) served as control. (c), The WB analysis of p-AKT and p-ERK in 786-O, 786-mut1, 786-mut2, A-704, A-704-mut1, and A-704-mut2 cells. AKT and ERK served as control. (d), The WB analysis of CTNNB1, p-GSK3ß, GSK3ß in 786-O, 786-mut1, 786-mut2, A-704, A-704-mut1, and A-704-mut2 cells. (e), The WB analysis of AKT, pAKT, pGSK3ß, GSK3ß, and CTNNB1 in 786-mut1 shCtrl, 786-mut1 shAKT, A-704-mut2 sh Ctrl, and A-704-mut2 shAKT cells. The RT-PCR analysis of AKT in 786-mut1 shCtrl, 786-mut1 shAKT, A-704-mut2 shCtrl, and A-704-mut2 shAKT cells. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01.

Fig 3: β-catenin is frequently altered in HCC patient samples(B) Frequency of genetic alteration in four genes, CTNNB1, APC, AXIN1 and FGF19, were determined in 207 HCC patients from ASAN collection (A) and 373 HCC patients from TCGA collection (B). Red indicates presence of genetic alteration and gray indicates wild-type. (C) Representative immunohistochemical analysis of normal liver and HCC. (D) Boxed plot of β-catenin cytoplasmic and nuclear staining (H-score) levels in human normal liver and HCC.

Fig 4: Haploid knockout of PIK3R1 promotes cancer stem cell phenotype through activating CTNNB1.(a), The representative photographs of cell spheres formed by 786-O, 786-mut1, 786-mut2, A-704, A-704-mut1, and A-704-mut2 cells. Spheres were calculated after two weeks cultivation at five independent fields/well. Scale bar = 50 µm. (b), 786-O and A-704 WT cells comprise fewer CD44+, CD133+, and CXCR4+ subset, respectively, compared with 786-O and A-704 WT cells. (c), The percentage of tumor-free mice 1 month after the subcutaneous injection of the different dilutions of 786-O, 786-mut1, 786-mut2, A-704, A-704-mut1, and A-704-mut2 cells into immunodeficient mice. The estimated percentage of renal cancer stem cells (RCSCs) in 786-O WT, 786-O Mut, A-704 WT, and A-704 Mut after the first transplantation using the extreme limiting dilution analysis. * P < 0.05, ** P < 0.01.

Fig 5: Effects of ESR2-selective antagonist PHTPP on the expression of non-phosphorylated CTNNB induced by DPN in the Sertoli cells from 20-day-old rats. Sertoli cells from 20-day-old rats were incubated in the absence (C, control) and presence of DPN (10 nM) for 24 h. The cells were also untreated or pretreated with ESR2-selective antagonist PHTPP (10 nM) for 30 min. Afterward, the cells were stimulated with DPN (10 nM) 24 h. Immunostaining for CTNNB (green) was detected using an antibody specific for non-phosphorylated CTNNB and Alexa Fluor 488-labeled secondary antibody. Nuclei were stained with DAPI (blue). Negative control (NC) was performed using normal rabbit serum at the same dilution of antibody. Scale bar as indicated. The data shown are representative of three independent experiments.