mr short hairpin rna shrna  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: Molecular interactions of glucocorticoid and mineralocorticoid receptors define novel transcription and biological functions

doi: 10.1016/j.jbc.2025.108488

Figure Lengend Snippet: MR coexpression suppresses DEX-induced apoptosis in GR-expressing cells. A , heat map showing diminished modulation of DEX-regulated genes related to apoptosis in MR/GR cells compared to those in GR cells. B , DEX induced cell death in GR cell, MR cell, MR/GR cell, and MR siRNA-treated MR/GR (MR/GR MR-KD) cells. MR coexpression suppressed the DEX-induced apoptosis in GR cells and apoptosis re-emerged when MR expression was suppressed. Cells were incubated with 100 nM DEX for 48 h. C , FACS quantification of cell death data shown in ( B ). D , MR mRNA and protein expressions in cell lines used for cell death analyses in ( B ). siRNA treatment of MR/GR cells (MR/GR MR-KD) effectively suppressed MR expression compared with control siRNA treatment (MR/GR NTC). E , propidium iodide negative and annexin-V positive cells quantification of cell lines treated with DEX as in ( B ) suggested cell death was caused by apoptotic mechanisms. F , PARP cleavage in the cells used in ( B ) double confirmed apoptosis is causing cell death observed in ( B ). Cleaved PARP and PARP amounts were determined by Western blotting as shown in the right panel . All quantified data in ( B ), ( D ), ( E ), and ( F ) are presented as mean ± SD with individual data points of technical replicates. The data were analyzed using one-way ANOVA with Sidak’s post hoc test. ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. DEX, dexamethasone; GR, glucocorticoid receptor; MR, mineralocorticoid receptor.

Article Snippet: The cells were cultured in a serum-free medium and transfected with 30 nM nontarget control siRNA or MR siRNA (SMART pool, Thermo Fisher Scientific) using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, Control, Western Blot

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