Fig. 5: ROS promotes PGAM5 cleavage and release into the cytoplasm for binding to MST3.
a Western blotting for PGAM5, MST3 and pMST3T178 in NCM460 cells after treatment with different concentrations of CCCP (0 µM, 10 µM, 20 µM, 30 µM) for 2 h. β-Actin was used as the loading control. b Western blotting was used to detect cytoplasmic and mitochondrial PGAM5 in NCM460 cells treated with 20 µM CCCP for 2 h. TOM20 was used as the mitochondrial positive control, β-Actin was used as the cytoplasmic positive control. c Co-IP assay of endogenous interaction between PGAM5 and MST3 in NCM460 cells treated with 20 µM CCCP for 2 h. β-Actin was used as the loading control. d Fluorescence intensity of MitoSOX (red) was detected with mitochondrial reactive oxygen species (mtROS) fluorescence probe. NCM460 cells were treated with 20 µM CCCP or 30 µM rotenone with or without 100 µM NAC for 2 h. n = 3 independent samples. Scale bar: 300 µm. e, f NCM460 cells were treated with 20 µM CCCP or 30 µM rotenone with or without 100 µM NAC for 2 h, MitoSOX fluorescence intensity was quantified using a microplate reader. n = 4 independent samples. P < 0.001. g, h Western blotting for PGAM5, MST3, pMST3T178, YAP and pYAPS127 in NCM460 cells treated with 20 µM CCCP (g) or 30 µM rotenone (h) with or without 100 µM NAC for 2 h. β-Actin was used as the loading control. i Immunofluorescence for GFP-Parkin (green) and mitochondrial targeting probe (red) was examined after NCM460 cells were treated with 30 µM rotenone with or without 100 µM NAC for 2 h. n = 3 independent samples. Scale bar: 25 µm. j Western blotting for PGAM5, PARL, MST3, pMST3T178, YAP and pYAPS127 in NCM460 cells transfected with siPARL for 24 h and treated with 30 µM rotenone for 2 h. α-Tubulin was used as the loading control. k Gross images of xenografted tumors 3 weeks after transplantation of siPGAM5 and/or pcDNA3.1-PARL transfected HCT116 cells. Quantification of tumor weight was performed. n = 6 mice. P = 0.0002; P = 0.0001; P < 0.001. l Western blotting for PARL, PGAM5, MST3, pMST3T178, YAP and pYAPS127 in HCT116 cells transfected with siPGAM5 and/or pcDNA3.1-PARL. β-Actin was used as the loading control. 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 < 0.001. Statistical analysis in panels e, f and k was performed with one-way ANOVA followed by Tukey’s test.
