Fig. 5: pVHL protects TFAM from LONP1 degradation.
a,b, Immunoblot analysis of primary Egln3+/+ and Egln3−/− MEFs (a) and 786-O cells (b) treated with 1 μM LONP1 inhibitor bortezomib (BTZ) for 16 h. c, Immunoblot analysis of HEK293 cells transfected with transposon vectors pB-TRE-TFAM-WT-Luc2, pB-TRE-TFAM-mut-Luc2 and transposase vector pCAG-hyPBase. d, Immunoblot analysis of PKA activity assay using biotinylated TFAM peptides with double hydroxyl-prolines 53 and 66. e,f, Immunoblot analysis of 786-O cells treated with 20 μM PKA activator forskolin (24 h) (e) or 5 μM PKA inhibitor H89 (24 h) (f). g, Immunoblot analysis of TFAM degradation assay by LONP1 using purified His-TFAM, GST-VHL, LONP1 and IVT-synthesized Flag-EGLN3 WT or Flag-EGLN3 catalytic mutant. In a–g, n = 3 biological independent experiments. h,i, Kaplan–Meier overall survival curve for individuals with high (blue) and low (red) expression of PKA catalytic subunit (PRKACA) (h) and LONP1 (i) using the Kidney Renal Clear Cell Carcinoma dataset from The Cancer Genome Atlas which contains 533 tumour samples (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi; minimal patient group size of 50 in the iterations). The overall survival probability was estimated with the KaplanScanner tool, using a Bonferroni-corrected logrank test between the two groups of patients. The graph depicts the best P value corrected for multiple testing (Bonferroni method). j, Crystal violet staining of 786-O cells pretreated with BTZ (10 nM) for 24 h and then treated for 48 h with sorafenib (Sora; 20 μM), BTZ (10 nM) or a combination (combo) of these two drugs as indicated. k, Cell apoptosis rate was detected by Annexin V-FITC/propidium iodide (PI) staining using flow cytometry. Data are presented as mean values ± s.d. Two-way ANOVA Tukey’s multiple-comparison test. ****P < 0.0001. n = 3 biological independent experiments. l, Female athymic NCrnu/nu mice were implanted subcutaneously with 786-O cells. Sorafenib (n = 4) or vehicle control (DMSO, n = 5) was administered orally, once a day at the dose of 15 mg per kg body weight. BTZ (n = 5) was administered by intraperitoneal injection, twice per week at a dose of 1 mg per kg body weight. Combined treatment: 1 mg per kg body weight BTZ + 15 mg per kg body weight sorafenib (n = 5). Mean (±s.e.m.) tumour volume data are shown. *P < 0.01, **P < 0.01, ***P < 0.001. m, Representative images of tumours after dissection and quantification of tumour weight of each treatment group. n, Representative H&E (scale bar indicates 50 µm, ×100), TFAM and MTCO2 immunofluorescence stainings (scale bar indicates 50 µm, ×400) of tumour tissues including quantification. NS, not significant.
