Journal: Biomedicines

Article Title: Aldosterone Suppresses Endothelial Mitochondria through Mineralocorticoid Receptor/Mitochondrial Reactive Oxygen Species Pathway

doi: 10.3390/biomedicines10051119

Figure Lengend Snippet: Aldosterone inhibits mitochondrial DNA in a dose-dependent and time-dependent manner through the MR pathway in HUVECs. ( A ) The mitochondrial DNA Cyt b copy number and ( B ) the mitochondrial DNA COII copy number were quantified by qPCR in HUVECs treated with different aldosterone concentrations (10 −10 , 10 −9 , 10 −8 , 10 −7 M) for 72 h. ( C ) The mitochondrial DNA Cyt b copy number and ( D ) the mitochondrial DNA COII copy number were quantified by qPCR at 24, 48, and 72 h in HUVECs treated with 10 −7 M aldosterone. ( E ) The mitochondrial DNA Cyt b copy number and ( F ) the mitochondrial DNA COII copy number were quantified by qPCR in HUVECs at 72 h after transfection of the HUVECs with GR siRNA (50 nM) or MR siRNA (100 nM) for 6 h before 10 −7 M aldosterone treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, compared between two groups using the t -test.

Article Snippet: GR siRNA (sc-35505), MR siRNA (sc-38836), and its scramble (Scr) control (sc-37007) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). siRNA Transfection Reagent (sc-29528) (Santa Cruz, CA, USA) was used as a lipid reagent for transfection.

Techniques: Transfection

Journal: Biomedicines

Article Title: Aldosterone Suppresses Endothelial Mitochondria through Mineralocorticoid Receptor/Mitochondrial Reactive Oxygen Species Pathway

doi: 10.3390/biomedicines10051119

Figure Lengend Snippet: Aldosterone inhibits mitochondrial and SOD2 protein expression in a dose-dependent and time-dependent manner through the MR pathway in HUVECs. ( A ) Western blot analysis of mitochondrial and SOD2 protein in HUVECs treated with different aldosterone concentrations (10 −10 , 10 −9 , 10 −8 , 10 −7 M) for 72 h. ( B ) Relative mitochondrial and SOD2 protein ratios are indicated in GAPDH using Image J software in HUVECs treated with different aldosterone concentrations (10 −10 , 10 −9 , 10 −8 , 10 −7 M) for 72 h. ( C ) Western blot analysis of the mitochondrial and SOD2 protein in HUVECs treated with 10 −7 M aldosterone for 24, 48, and 72 h. ( D ) Relative mitochondrial and SOD2 protein ratios are indicated in GAPDH using Image J software in HUVECs treated with 10 −7 M aldosterone for 24, 48, and 72 h. ( E ) HUVECs were treated with different concentrations of aldosterone (10 −10 , 10 −9 , 10 −8 , 10 −7 M) and vehicle (equal volume of DMSO) for 72 h. Mitochondria were stained red with anti-mitochondrial antibodies, and cell nuclei were stained blue with DAPI. Quantitative fluorescence intensity was analyzed using Image J software. ( F ) HUVECs were transfected with GR siRNA (50 nM) or MR siRNA (100 nM) for 6 h before 10 −7 M aldosterone treatment. After 72 h, mitochondria were stained red with anti-mitochondrial antibodies, and cell nuclei were stained blue with DAPI. Representative images were captured using a fluorescence microscope with 40x magnification. Scale bar: 100 μm. Quantitative fluorescence intensity was analyzed using Image J software. * p < 0.05, compared between two groups using the t -test.

Article Snippet: GR siRNA (sc-35505), MR siRNA (sc-38836), and its scramble (Scr) control (sc-37007) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). siRNA Transfection Reagent (sc-29528) (Santa Cruz, CA, USA) was used as a lipid reagent for transfection.

Techniques: Expressing, Western Blot, Software, Staining, Fluorescence, Transfection, Microscopy

Journal: Biomedicines

Article Title: Aldosterone Suppresses Endothelial Mitochondria through Mineralocorticoid Receptor/Mitochondrial Reactive Oxygen Species Pathway

doi: 10.3390/biomedicines10051119

Figure Lengend Snippet: Mito-TEMPO reduces the function of mitochondrial in the aldosterone-stimulated HUVECs. HUVECs were treated with Mito-TEMPO (10 uM) for 2 h before being treated with 10 −7 M aldosterone. ( A ) After 24 h, immunofluorescence images showed mtROS production. ( B ) HUVECs were transfected with GR siRNA (50 nM) or MR siRNA (100 nM) for 6 h and then treated with 10 −7 M aldosterone. MtROS was stained red with MitoSox, and mitochondria were stained green with MitoTracker. ( C ) The mitochondrial DNA Cyt b copy number (up) and mitochondrial DNA COII copy number (down) were quantified by qPCR. ( D ) Mitochondria were stained red with anti-mitochondrial antibodies, and cell nuclei were stained blue with DAPI. Representative images were captured using a fluorescence microscope with 40x magnification. Scale bar: 100 μm. Quantitative fluorescence intensity was analyzed using Image J software. * p < 0.05, ** p < 0.01, compared between two groups using the t -test.

Article Snippet: GR siRNA (sc-35505), MR siRNA (sc-38836), and its scramble (Scr) control (sc-37007) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). siRNA Transfection Reagent (sc-29528) (Santa Cruz, CA, USA) was used as a lipid reagent for transfection.

Techniques: Immunofluorescence, Transfection, Staining, Fluorescence, Microscopy, Software