Journal: Cell proliferation

Article Title: SIRT1 Alleviates Oxidative Stress-Induced Mitochondrial Dysfunction and Mitochondria-Associated Membrane Dysregulation in Stress Urinary Incontinence.

doi: 10.1111/cpr.70009

Figure Lengend Snippet: FIGURE 8 | The effects of SIRT1 upregulation on structural and functional alterations of MAMs and calcium homeostasis in L929 cells under oxi- dative stress. (A) Immunoblot analyses of PERK and GRP78 in L929 cells. Quantification represents the levels of the indicated proteins normalised to β-Actin. (B) Representative TEM images of mitochondrial ultrastructure of L929 cells. Blue arrows indicated MAMs. Quantitative analysis of MAM parameters, including MAM distance and length to mitochondrion in TEM images. (C) Immunoblot analyses of SERCA2, GRP75, MCU and VDAC1 in L929 cells. Quantification represents the levels of the indicated proteins normalised to β-Actin. (D) Co-localization analysis of immunofluores- cence images of IP3R (green) and MitoTracker (red) in different groups of L929 cells. (E) Co-localization fluorescence imaging was conducted with MitoTracker, demonstrating that Rhod-2 AM is predominantly an indicator of mitochondrial Ca2+ levels. (F) Concentration of mitochondrial calcium ions. (G) Representative flow plots of mitochondrial ROS production in L929 cells measured by flow cytometry. Data are expressed as mean ± SD. *p < 0.05 compared with control group, #p < 0.05 compared with H2O2 group.

Article Snippet: After being blocked with 5% skim milk for 1 h, the membranes were incubated overnight at 4°C with primary antibodies against SIRT1 (1:1000, Proteintech Group Inc.), PGC- 1α (1:1000, Proteintech Group Inc.), NRF1 (1:5000 Proteintech Group Inc.), TFAM (1:5000, Proteintech Group Inc.), PINK1 (1:2000, Proteintech Group Inc.), Parkin (1:2000, Proteintech Group Inc.), Collagen I (1:1000, Proteintech Group Inc.), Collagen III (1:1000, Proteintech Group Inc.), Cytochrome c (1:1000, Proteintech Group Inc.), VDAC1 (1:2000, Proteintech Group Inc.), GRP75 (1:5000, Proteintech Group Inc.), FUNDC1 (1:1000, Cell Signalling Technology Inc.), PERK (1:1000, Proteintech Group Inc.), GRP78 (1:3000, Proteintech Group Inc.), SERCA2 (1:5000, Proteintech Group Inc.), MCU (1:3000, Proteintech Group Inc.) and β- actin (1:20,000, Proteintech).

Techniques: Functional Assay, Western Blot, Fluorescence, Imaging, Concentration Assay, Flow Cytometry, Control