Fig. 7: PGAM5 regulates YAP activity and CRC growth through MST3.

a Representative image of AOM-DSS tumors and quantification of tumor numbers per mouse in Ctrl, Mst3^cKO (P = 0.026), Pgam5^cKO (P = 0.002) and dKO (P = 0.022) mice. n = 3. b Western blotting for Mst3, pMst3^T178, Pgam5, Yap, pYap^S112, Lats1, pLats1^T1079 in colon tumors from Ctrl, Mst3^cKO, Pgam5^cKO and dKO mice. β-Actin was used as the loading control. c-e Double immunofluorescence of Ki67/β-Catenin or Yap/E-cadherin in colon tumors of Ctrl, Mst3^cKO, Pgam5^cKO and dKO mice (e). The percentage of Ki67⁺ cells (c, n = 31 crypts from 3 mice. P = 0.003; P = 0.0001) and nuclear YAP⁺ cells (d, n = 37 crypts from 3 mice) in tumors were quantified. Scale bar: 100 µm. f Gross images of xenograft tumors 3 weeks after transplantation of shMST3- and/or shPGAM5-transfected HCT116 cells. n = 6. g Growth of APKS mouse tumor organoids after transfection with siMST3 and/or siPGAM5. Organoid areas were quantified at the indicated time points. 12 h, n = 46 fields from 3 independent samples; 96 h, n = 43 fields from 3 independent samples. Scale bar: 250 µm. P < 0.001; P < 0.001; P = 0.024; P < 0.001. h Western blotting for MST3, PGAM5 and Flag in HEK293FT cells transfected with siMST3 and/or siPGAM5 and/or flag-YAP. β-Actin was used as the loading control. i Luciferase assay for TEAD luciferase reporter genes in HEK293FT cells transfected with flag-YAP, followed by siMST3 and/or siPGAM5, respectively. n = 6 independent samples. j Western blotting for MST3 and PGAM5 in HCT116 cells transfected with pcDNA3.1-MST3 and/or pcDNA3.1-PGAM5 plasmids. β-Actin was used as the loading control. k qRT-PCR for YAP, CTGF (P = 0.002; P = 0.021) and CYR61 (P = 0.018; P = 0.025) in HCT116 cells transfected with pcDNA3.1-MST3 and/or pcDNA3.1-PGAM5 plasmids. n = 3 independent samples. Western blotting images are representative of experiments that were repeated at least three times with similar results. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Statistical analysis in panels (a, g, and i) was performed with one-way ANOVA followed by Tukey’s test, and the rest were performed with two-tailed unpaired Student’s t-test.