Fig 2: BRCA1 knockdown represses breast cancer cell growth and lower BRCA1 expression correlates with longer relapse-free survival rate. (a) MCF7 cells stably expressing Tet-shRNA vector against BRCA1 (Tet-shBRCA1) or control vector (Tet-shNT) were cultured with or without 0.1 µg/ml DOX for 48 h. BRCA1 knockdown efficiency was confirmed by Western blotting. (b) Growth of MCF7 cells expressing the Tet-shNT, Tet-shBRCA1 #1, or #3 vectors was assessed using the IncuCyte live-cell imaging system in the presence or absence of 0.1 µg/ml DOX for 4 days. n = 4, means ± S.D. *p < 0.05 versus -DOX. (c) Comparison of colony formation ability between MCF7 cells expressing Tet-shNT and Tet-shBRCA1. Cells were seeded in 6-well plates and cultured for 3 weeks in the presence or absence of 0.1 µg/ml DOX. Visualized colonies are shown in the left panel. Summarized colony numbers are shown in the right panel. Bar = means ± S.D. n = 3, *p < 0.05 versus -DOX. (d) The growth of PINK1 knockout MCF7 cells (sgPINK1#1, #2) or control cells (sgNega3, 5) was assessed using the IncuCyte live-cell imaging system. n = 5, means ± S.D. *p < 0.05 versus sgNega5. (e) Relapse-free survival rates of breast cancer patients were compared between high and low BRCA1, Parkin, or PINK1 expression in patients. Data are shown as Kaplan–Meier plots; graphs were generated using an online Kaplan–Meier plotter.

Fig 3: miR-593-5p directly targets PINK1 gene.A, SH-SY5Y transfected with miR-593-5p or miR-SC were treated with CCCP (10 µM), MG-132 (10 µM), or both for 2 h. Western blots were performed using Pink1 and ß-actin antibodies. B, the band intensities of 65 kDa and 50 kDa Pink1 were measured from three independent Western blot experiments and normalized to those of ß-actin. Statistical significance was determined by ANOVA followed by Bonferroni’s post hoc test. Data are mean ± SD for three biological replicates. t test, **p < 0.01. C, reporter assay using luciferase construct containing PINK1-3'UTR or synthetic miR-593-5p's target sequence. SH-SY5Y cells were cotransfected as indicated along with pSV-ß-galactosidase (Promega). Luciferase activity was normalized against ß-galactosidase activity. D, determination of PINK1 mRNA levels in SH-SY5Y cells transfected with miR-593-5p or miR-SC by qRT-PCR. Data (C and D) are mean ± SD for three biological replicates. Each circle represents the mean of three technical replicates. t test, **p < 0.01, ns, nonsignificant. CCCP, carbonyl cyanide m-chlorophenylhydrazone; PINK1, PTEN-induced putative kinase 1.

Fig 4: miR-593-5p targets the CDS of PINK1 mRNA.A, schematic drawings illustrating the potential target sites of miR-593-5p in the CDS of PINK1 mRNA and potential base-pairings between miR-593-5p and target sites (I, II, and III). Watson-Crick base pairs are represented in red and wobble base pairs (G:U) are represented in blue. B and C, SH-SY5Y cells were transfected as indicated. Western blots were performed with anti-FLAG and anti-ß-actin. D, band densities of PINK1-flag were measured and normalized to those of ß-actin. Band densities were calculated from three independent Western blot experiments of C. Data are mean ± SD for three biological replicates. E, SH-SY5Y cells were transfected as indicated along with pSV-ß-galactosidase. Luciferase activities were measured and normalized to ß-galactosidase activity. Data are mean ± SD for three biological replicates. Each circle represents the mean of three technical replicates. t test, *p < 0.05. CDS, coding sequence; PINK1, PTEN-induced putative kinase 1.

Fig 5: The PINK1 p.I368N mutation is positioned in the kinase domain and alters the ATP-binding site. a Domain structure of human PINK1 protein. Individual domains are color-coded and labeled: mitochondrial targeting sequence (MTS); transmembrane domain (TM); N-terminal, regulatory domain (NT); N-lobe and C-lobe of the kinase domain; C-terminal domain (CTD). Residues surrounding Ile368 were aligned with corresponding sequences of PINK1 orthologs from different species. Horizontal bar denotes the catalytic loop in kinase subdomain VIb. b Root mean square fluctuations (RMSF) of the entire PINK1 structures. Dotted lines separate individual domains of PINK1 that include MTS, TM, NT, kinase domain and CTD. An arrow highlights the position of the p.I368N mutation. c Superposition overlay of the kinase domains before and after 50 ns of MDS. Structures are shown in cartoon ribbons with PINK1 WT in gray and p.I368N mutant in color according to the domain structure in (a). Residues Ile368 and Asn368 are shown in licorice rendering using gray or standard atom coloring, respectively. The Mg2+ ions are gray colored Van der Waals (VdW) spheres for both and the ATP-analogs in the active sites are shown in gray for WT and Goodsell coloring for p.I368N. d Root mean square deviation (RMSD) of the kinase domain of PINK1 WT and p.I368N mutant, shows only minor structural differences. e Zoom into the ATP-binding sites of PINK1 WT and p.I368N mutant in superposition overlay before and after 50 ns of MDS as described for (c). f RMSD of the ATP-binding pocket in the kinase domains of WT and mutant PINK1. Pocket residues include positions: 161,163, 217, 218, 219, 237, 240, 275, 316, 318, 319, 320, 321, 322, 325, 326, 327, 362, 363, 365, 366, 367, 368, 369, 383, 384, 385, 386, and 387. g The surface of the ATP-binding pocket of WT and p.I368N mutant PINK1 is shown for comparison in standard atom coloring. The Mg2+ ion (VdW sphere) and the ATP-analog (sticks colored by atom type) in the active site are given. h Radius of gyration measurements on the ATP-binding pocket from WT and mutant PINK1 provides the extent to which an object is extended from its center of mass