Fig 2: PolG D257A mutation attenuates the stabilizing effect of p53-PolG complex on mitochondria and aggravates vascular calcification. The WT mice and POLGD257A/D257A mice was treated with or without 8*10^5 UI/Kg Vit D3 respectively to construct calcification model. (A) Von-Kossa staining of mice aortas. Black arrows pointed to deposition of calcium or calcified VSMCs, and hollow arrows pointed to the normal or pathology structure of vessels. (B) Calcium deposition of aortas, data were collected from 10 individual mice (10 mice in one experiment group) and expressed as the mean ± SD. ***p < .001. (C) MBP of mice in different treatment groups (n=10). Data were expressed as the mean ± SD. ***p < .001. (D, E) Sacrificed mice and isolated the aortas, then conducted WB assay to detected expression of Runx 2, BMP 2 and Tublin, Tublin was set as a loading control. Data were collected from 10 individual mice and expressed as the mean ± SD. ***p < .001. (F, G) Sacrificed mice and isolated the aortas, then extracted mitochondria from them. Conducted WB 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 10 individual mice and expressed as the mean ± SD. the negative control was set as the panel which were not treated with Vit D3 in each location and mice type, #p < .05. *p < .05, ns p>0.05. (H, I) 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.

Fig 3: 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 4: Mutant PolG is fail to stabilize mitochondrial function and against calcification. VSMCs was transfected with Myc-vector, WT PolG and mutant PolG with or without 10mmol β-GP induced calcification 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 < .001, 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, ns p>0.05. (D) ATP contents of VSMCs. Data were collected from 3 repeat experiment and expressed as the mean ± SD. ***p < .001, ns p>0.05. (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, ns p>0.05. (G, H) Western blot analysis and quantification of protein expression (Myc-tag, Runx 2, BMP 2 and GAPDH), GAPDH was used as a loading control, and the VSMCs transfected with Myc-tag vector and without treated with β-GP was set as control in each parameter. ***p < .001. Data were expressed as the mean ± SD of triplicate experiments.

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.