Fig 2: NEK10 depleted cells show a decrease in mitochondrial mass. Expression of four different mitochondrial marker proteins (a TOM20, b TFAM, c VDAC and d OXPHOS) was analyzed from HeLa pLKO, HeLa pLKO-sh89 and HeLa pLKO-sh90 total lysates. For immunoblotting, the following antibodies were used: anti-TOMM20, anti-mtTFAM, anti-VDAC1 and anti-OXPHOS. The quantification graphs on the right show averages from 4 independent experiments. The statistical analyses were performed using One-Way ANOVA followed by Tukey’s Multiple Comparison Test. (* = P < 0.05; ** = P < 0.01; *** = P < 0.001)

Fig 3: NEK10 is localized in the mitochondria. a Mitochondrial fractionation assay. Mitochondria from HeLa pLKO, HeLa pLKO-sh89 and HeLa pLKO-sh90 cells were isolated and NEK10 was analyzed by Western blot using anti-NEK10 antibody. Postnuclear supernatant (PNS), cytosol (CYT) and mitochondrial (MITO) fractions were analyzed with anti-Tubulin B, anti-OXPHOS and anti-VDAC antibodies, for fraction purity. The letter A and the red arrows indicate the 133 kDa isoform, present in PNS and CYTO. The letter B and the blue arrows indicate the 80 kDa isoform, present in the mitochondrial fraction (MITO). b Confocal images of NEK10 in mitochondria. HeLa pLKO-empty cells were stained using anti-NEK10 antibody (green) and the mitochondria were visualized with MitoTracker DeepRed for 20 min (red). Cells were analyzed in a confocal Zeiss LMS 510 microscope, using 63x oil objective, 1 air unit pinhole, 2048 × 2048 pixels. Scale bars: 10 µm. Images were analyzed using FIJI software [26]. Insets are enlargements of the outlined areas

Fig 4: Mitochondrial DNA (mtDNA) analysis of HeLa pLKO-empty, HeLa pLKO-sh89 and HeLa pLKO-sh90 cells. a The mtDNA integrity of NEK10 depleted and control cells was analyzed by Long-Extension PCR and the ratio of the amplification is shown in the graph in basal conditions. b The mtDNA integrity of cells treated with 300 µg/ml zeocin treatment for 3 h was evaluated by long-extension PCR. Results show the difference in mtDNA damage of cells with zeocin treatment from the mtDNA damage under basal conditions. The statistical analysis is a comparison to the control wild type cells. c The number of mitochondrial DNA copy number by the different cells analyzed by Real-Time quantitative polymerase chain reaction (qPCR). Both graphs show analyses from n = 3 independent experiments. Statistical analyses were made by One-Way ANOVA and the Tukey’s Multiple Comparison Test. * = P < 0.05; ** = P < 0.01

Fig 5: NEK10 interacts with mitochondrial protein partners. Interaction network of human NEK10 with partners identified by IP-LC-MS/MS reveals mitochondrial proteins enrichment after zeocin treatment. The proteomic data retrieved from IP-LC-MS/MS was submitted to the Integrated Interactome System (IIS) platform (National Laboratory of Biosciences, Campinas, Brazil) [22]. The protein-protein interaction network (PPI) was generated using Cytoscape software [24]. a The fold change in the number of the proteins found after zeocin treatment, relative to before treatment, for the main cellular compartments enriched in the network is shown in the graph. ER = endoplasmic reticulum, b Protein network showing mitochondrial proteins found in samples without treatment (purple nodes) and with zeocin treatment (red nodes). Clusters were made according to the Selected Cellular Component. SIRT3 is a protein interactor retrieved from the databank by IIS platform [22]. c Table showing mitochondrial proteins found in untreated and zeocin treated samples