Journal: International Journal of Biological Sciences

Article Title: DNA Polymerase Gamma Acetylation Governs Mitochondrial Homeostasis and Vascular Cell Senescence

doi: 10.7150/ijbs.122298

Figure Lengend Snippet: D257A mutation exacerbates senescence via dysregulated HASMC function and mitochondrial dysfunction. (A) The different expression of senescence marker in the aorta of POLG WT/WT mice and POLG D257A/D257A mice by WB assay, the right panel is the quantification analysis. WT is for POLG WT/WT mice and HO is for POLG D257A/D257A mice. The expression of GAPDH was used as a loading control. ***p < .001. ## p < .01, # p < .05; (B) SA-β-Gal staining for MEFs with different genotype and passage, the rate of positive staining cells was counted to analysis. ***p < .001, *p < .05; (C) Co-IP analysis and quantification analysis of relative acetylation level of Polγ in aorta of different genotype and age. ***p < .001. ## p < .01; (D-E) The collagen gel contraction experiment of primary HASMCs with different genotype and passage. The percentage of gel surface area contraction (%) was calculated using the formula: [(0h area - 12h area) / 0h area] × 100%. ***p < .001, *p < .05; (F) The expression of various Ca 2+ channel related proteins in aorta of POLG WT/WT mice and POLG D257A/D257A mice by WB assay, the median and right panels are quantification analysis. The expression of GAPDH was used as a loading control. ***p < .001, ### p < .001; (G) The cell viability experiment of primary HASMC of POLG WT/WT mice and POLG D257A/D257A mice by CCK-8 kit assay in 12 hours, 24 hours and 36 hours. ***p < .001, *p < .05; (H) The colony forming experiment of primary HASMC of POLG WT/WT mice and POLG D257A/D257A mice, the cells were cultured for 7 days, staining and counting the colony numbers in 1day, 3days and 7days. Counting the colony numbers to analysis. ***p < .001, *p < .05, ns p>0.05; (I) The cell viability experiment of primary HASMC of POLG WT/WT mice and POLG D257A/D257A mice with different passage by CCK-8 kit assay in 12 hours, 24 hours and 36 hours. ***p < .001; (J) The JC-10 measurement of MMP of the primary HASMC of different genotype and passage by flow cytometry. Compare the difference of monomeric cells ratio (green) in every subgroup. ***p < .001, *p < .05; (K) The expression of metabolic proteins (citrate synthase, p-PKM2, PKM2) in aorta of different genotype and age. The expression of Tubulin was used as a loading control for citrate synthase, and PKM2 was used as a loading control for p-PKM2. The median and right panels are quantification analysis, ***p < .001, **p < .01, *p < .05; (K) Schematic diagram. All data were expressed as the mean ± SD of triplicate experiments. In each independent experiment, the sample size for each group of experiments is 3.

Article Snippet: Acetylated-lysine, Cell Signaling Technology (USA) Cat #9441; Citrate Synthase, Santa Cruz (USA) Cat #sc-6246; COX IV, Wanleibio (China) Cat #WL01418; Flag-tag, Absmart (China) Cat #M20008; Flag-tag, GNI (Japan) Cat #GNI4110-FG-M; GAPDH, Proteintech (China) Cat #60004-1-Ig; GCN5, Proteintech (China) Cat #66575-1-Ig; HRP goat anti-mouse IgG, ABclonal Biotech Cat #AS003; HRP goat anti-rabbit IgG, ABclonal Biotech Cat #AS014; L-type Ca 2+ CP, Santa Cruz (USA) Cat #sc-515679; Myc-tag, Cell Signaling Technology (USA) Cat #2276; p16, Proteintech (China) Cat #10883-1-AP; p16, Wanleibio (China) Cat #WL02203; p21, Santa Cruz (USA) Cat #sc-6246; PKM2, Cell Signaling Technology (USA) Cat #4053S; PKM2-phosphorylation, Cell Signaling Technology (USA) Cat #3827S; Polγ, Abcam (USA) Cat # ab128899; Polγ, Abcam (USA) Cat # ab97661; Polγ, Cell Signaling Technology (USA) Cat #13609; Polγ, Santa Cruz (USA) Cat #sc-390634; Sirt3, Santa Cruz (USA) Cat #sc-6246; TRPC6, Santa Cruz (USA) Cat #sc-515837; Tubulin, Proteintech (China) Cat #11224-1-AP.

Techniques: Mutagenesis, Expressing, Marker, Control, Staining, Co-Immunoprecipitation Assay, CCK-8 Assay, Cell Culture, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: DNA Polymerase Gamma Acetylation Governs Mitochondrial Homeostasis and Vascular Cell Senescence

doi: 10.7150/ijbs.122298

Figure Lengend Snippet: Acetylated Polγ regulates HASMC function and mitochondrial homeostasis via mtDNA binding. (A) Evaluate the effect of different acetylated Polγ in contractive ability by collagen gel contraction experiment. The percentage of gel surface area contraction (%) was calculated using the formula: [(0h area - 12h area) / 0h area] × 100%. ***p < .001, **p < .01, ### p < .001, ## p < .01, ns p>0.05; (B-C) Transfect the HASMCs with various acetylated Polγ and compare the expression of L-type Ca 2+ CP and TRPC6. The expression of Tubulin was used as a loading control. ***p < .001, ### p < .001, ## p < .01, ns p>0.05; (D) Compare the proliferation alteration of HASMCs after transfecting various acetylated Polγ by CCK-8 assay. ***p < .001, **p < .01, ### p < .001; (E-G) Conduct WB analysis and quantification analysis to evaluate protein expression (citrate synthase, p-PKM2, PKM2, Tubulin) in HASMC which were transfected with different acetylated Polγ. The expression of Tubulin was used as a loading control for citrate synthase, and PKM2 was used as a loading control for p-PKM2. ***p < .001, # p < .05, ns p>0.05; (H-I) The MMP alteration under various acetylated Polγ conditions by JC-10 assay. Compare the difference of monomeric cells ratio (green) in every subgroup. ***p < .001, *p < .05, # p < .05; (J) The binding degree between different acetylated Polγ and mtDNA by Ch-IP assay. ***p < .001, **p < .01; (K) Schematic diagram. All data were expressed as the mean ± SD of triplicate experiments. In each independent experiment, the sample size for each group of experiments is 3.

Article Snippet: Acetylated-lysine, Cell Signaling Technology (USA) Cat #9441; Citrate Synthase, Santa Cruz (USA) Cat #sc-6246; COX IV, Wanleibio (China) Cat #WL01418; Flag-tag, Absmart (China) Cat #M20008; Flag-tag, GNI (Japan) Cat #GNI4110-FG-M; GAPDH, Proteintech (China) Cat #60004-1-Ig; GCN5, Proteintech (China) Cat #66575-1-Ig; HRP goat anti-mouse IgG, ABclonal Biotech Cat #AS003; HRP goat anti-rabbit IgG, ABclonal Biotech Cat #AS014; L-type Ca 2+ CP, Santa Cruz (USA) Cat #sc-515679; Myc-tag, Cell Signaling Technology (USA) Cat #2276; p16, Proteintech (China) Cat #10883-1-AP; p16, Wanleibio (China) Cat #WL02203; p21, Santa Cruz (USA) Cat #sc-6246; PKM2, Cell Signaling Technology (USA) Cat #4053S; PKM2-phosphorylation, Cell Signaling Technology (USA) Cat #3827S; Polγ, Abcam (USA) Cat # ab128899; Polγ, Abcam (USA) Cat # ab97661; Polγ, Cell Signaling Technology (USA) Cat #13609; Polγ, Santa Cruz (USA) Cat #sc-390634; Sirt3, Santa Cruz (USA) Cat #sc-6246; TRPC6, Santa Cruz (USA) Cat #sc-515837; Tubulin, Proteintech (China) Cat #11224-1-AP.

Techniques: Binding Assay, Expressing, Control, CCK-8 Assay, Transfection

Journal: International Journal of Biological Sciences

Article Title: DNA Polymerase Gamma Acetylation Governs Mitochondrial Homeostasis and Vascular Cell Senescence

doi: 10.7150/ijbs.122298

Figure Lengend Snippet: K1039 acetylation status governs Polγ's role in metabolic and functional rescue. (A-B) Transfect Sh-Polγ-HASMCs with various acetylated Polγ condition plasmid, evaluating the contractive ability by collagen gel contraction experiment. The percentage of gel surface area contraction (%) was calculated using the formula: [(0h area - 12h area) / 0h area] × 100%. ***p < .001, **p < .01, *p < .01, ns p>0.05; (C-D) Knock-down the expression of Polγ in HASMCs and rescue with various acetylated Polγ and compare the expression of L-type Ca 2+ CP and TRPC6. The expression of Tubulin was used as a loading control. ***p < .001, **p < .01, *p < .01, ns p>0.05; (E) Evaluate the rescue effect of different acetylated Polγ in regulating the proliferation effect by CCK-8 assay. The results are defined as the absorbance in 450nm. ***p < .001, ns p>0.05; (F) Conduct WB analysis and quantification analysis to evaluate protein expression (citrate synthase, p-PKM2, PKM2, Tubulin) in Sh-Polγ-HASMC which were transfected with different acetylated Polγ plasmids for 48 hours. The expression of Tubulin was used as a loading control for citrate synthase, and PKM2 was used as a loading control for p-PKM2. ***p < .001, **p < .01, *p < .01, ns p>0.05; (G-H) The JC-10 assay to evaluate rescue effect of different acetylated Polγ in regulating MMP. Compare the difference of monomeric cells ratio (green) in every subgroup. **p < .01, *p < .01, ns p>0.05; (I). Schematic diagram. All data were expressed as the mean ± SD of triplicate experiments. In each independent experiment, the sample size for each group of experiments is 3.

Article Snippet: Acetylated-lysine, Cell Signaling Technology (USA) Cat #9441; Citrate Synthase, Santa Cruz (USA) Cat #sc-6246; COX IV, Wanleibio (China) Cat #WL01418; Flag-tag, Absmart (China) Cat #M20008; Flag-tag, GNI (Japan) Cat #GNI4110-FG-M; GAPDH, Proteintech (China) Cat #60004-1-Ig; GCN5, Proteintech (China) Cat #66575-1-Ig; HRP goat anti-mouse IgG, ABclonal Biotech Cat #AS003; HRP goat anti-rabbit IgG, ABclonal Biotech Cat #AS014; L-type Ca 2+ CP, Santa Cruz (USA) Cat #sc-515679; Myc-tag, Cell Signaling Technology (USA) Cat #2276; p16, Proteintech (China) Cat #10883-1-AP; p16, Wanleibio (China) Cat #WL02203; p21, Santa Cruz (USA) Cat #sc-6246; PKM2, Cell Signaling Technology (USA) Cat #4053S; PKM2-phosphorylation, Cell Signaling Technology (USA) Cat #3827S; Polγ, Abcam (USA) Cat # ab128899; Polγ, Abcam (USA) Cat # ab97661; Polγ, Cell Signaling Technology (USA) Cat #13609; Polγ, Santa Cruz (USA) Cat #sc-390634; Sirt3, Santa Cruz (USA) Cat #sc-6246; TRPC6, Santa Cruz (USA) Cat #sc-515837; Tubulin, Proteintech (China) Cat #11224-1-AP.

Techniques: Functional Assay, Plasmid Preparation, Knockdown, Expressing, Control, CCK-8 Assay, Transfection