Fig 2: p53-PolG D257A complex cannot respond to calcification stimulation to maintain mitochondrial function. (A, B) VSMCs were treated with 10mmol β-GP for 5 days, then harvested the cells and extracted mitochondria. Conducted Western blot assay to detected expression of p53, COX 4 and GAPDH. COX 4 was set as reference of mitochondrial proteins, and GAPDH was set as reference of cytoplasm proteins. Data were collected from 3 repeat experiment and expressed as the mean ± SD. ***p < .001. (C, D) CO-IP assay was conducted by using anti‐PolG or anti‐p53 antibody or negative control IgG and Protein A/G agarose beads to enrichment target proteins. The resulting precipitates were subjected to Western blot analysis with anti‐p53 or anti‐PolG. A portion of the whole-cell lysate was also subjected to WB analysis as input. (E, F) The VSMCs were treated with or without 10mmol β-GP for 5 days, then conducted CO-IP assay to detect interaction alteration of PolG and p53. CO-IP assay was conducted by using anti‐PolG or anti‐p53 antibody or negative control IgG and Protein A/G agarose beads to enrichment target proteins. The resulting precipitates were subjected to Western blot analysis with anti‐p53 or anti‐PolG. A portion of the whole-cell lysate was also subjected to WB analysis as input. (G, H) The VSMCs were transfected with Myc-vector, Myc-tag WT PolG or mutant PolG, treated the cells with or without 10mmol β-GP. Harvested the cells and conducted CO-IP assay to clarify interaction alteration of different types of PolG and p53. CO-IP assay was conducted by using anti‐PolG or anti‐p53 antibody or negative control IgG and Protein A/G agarose beads to enrichment target proteins. The resulting precipitates were subjected to Western blot analysis with anti‐p53 or anti‐PolG. A portion of the whole-cell lysate was also subjected to WB analysis as input.

Fig 3: Model for PolG rescues mitochondrial function and inhibits calcification by interacted with p53. PolG can recruit and interact with p53 to keep mtDNA stabilization, and these behaviors can be enhanced by calcification stimulation. While mutant PolG fails to form enhanced PolG-p53 complexes in response to calcification, eventually leading to mitochondrial dysfunction and severe calcification. See texts for details.

Fig 4: Waterfall plot of mtDNA changes in the siRNA screen. HeLa cells were grown in galactose medium and subjected to siRNA-mediated knockdown of the indicated candidate proteins associated with mitochondrial functions for 48 h. Cells were treated with DMSO (a) or 15 µM APE1inhIII (b) for 5 h. PCR products of the 7 kb long Set2 mtDNA region amplified from total DNA were quantified, normalized to the sample-specific values for the 0.2 kb short mitochondrial 12S region and plotted relative (rel.) to the mean values for nsRNA transfected samples per experiment (n = 6–8 from 4 independent experiments). All values are means ± SEM. Note that colored bars indicate data obtained after knockdown of high priority factors of this study further on, namely blue bars after ENDOG knockdown, green bar after POLGA knockdown, and orange bar after POLZ knockdown.

Fig 5: PolG deficiency induced aggravated calcification can be rescued by WT PolG but not the mutant PolG. NC VSMCs was transfected with Myc-vector and PolG knockdown VSMCs were transfected with Myc-vector, WT PolG or mutant PolG. All groups were treated with or without 10mmol β-GP for 5 days, and then conducted related experiment to detect mitochondrial function and calcification status. (A, B) Used flow cytometry to detect MMP level of mitochondria. The potential alteration was calculated as red/green fluorescent intensity ratio. Data were collected from 3 repeat experiment. **p < .005, ns p>0.05. (C) mtDNA copy numbers of VSMCs. Data were collected from 3 repeat experiment and expressed as the mean ± SD. ***p < .001, **p < .005. (D) ATP contents of VSMCs. Data were collected from 3 repeat experiment and expressed as the mean ± SD. ***p < .001, **p < .005. (E) Alizarin red staining of VSMCs. (F) Quantification of calcium deposition of VSMCs, data were collected from 3 repeat experiment and expressed as the mean ± SD. ***p < .001, **p < .005. (G, H) Western blot analysis and quantification of protein expression (Myc-tag, PolG, Runx 2, BMP 2 and GAPDH), GAPDH was used as a loading control, and the NCVSMCs transfected with Myc-tag vector and without treated with β-GP was set as control in each parameter. **p < .005. *p < .05, ns p>0.05. Data were expressed as the mean ± SD of triplicate experiments.