Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 and stromal-interacting molecule 1 (STIM1) expression in mouse pulmonary arterial smooth muscle cells (PASMCs). A: RT-PCR products from cultured mouse PASMCs amplified using primers for mouse Orai1 (269 bp), STIM1 (473 bp), and β-actin (498 bp). Three separate RT-PCR reactions were performed in the presence (+) and absence (−) of reverse transcriptase (RT). B: Orai1, STIM1, and GAPDH proteins were detected in cultured mouse PASMCs using Western blot analysis. Experiments were performed in 5 separate Western blot analyses.

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Amplification, Reverse Transcription, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: STIM1 is associated with Orai1 to mediate CCE in mouse PASMCs. A and B: STIM1, Orai1, and GAPDH proteins were detected in nontransfected mouse PASMCs and in PASMCs transfected with 200 nM scrambled siRNA (negative control). The expression of STIM1 but not Orai1 or GAPDH was reduced significantly in cells transfected with 200 nM STIM1 siRNA. The expressions of STIM1 and Orai1 but not GAPDH were reduced significantly in cells transfected with both 200 nM STIM1 siRNA and 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: siRNA knockdown of STIM1 reduced the CPA-induced transient and sustained increase in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. siRNA knockdown of STIM1 and Orai1 further reduced the CPA-induced transient but not sustained increase in fura-2 fluorescence ratio in the presence of nifedipine. D: bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in negative control cells (filled bars, n = 103), in STIM1 siRNA-transfected cells (shaded bars, n = 148), and in STIM1 siRNA and Orai1 siRNA-transfected cells (open bars, n = 70). **P < 0.01, compared with negative control cells (ANOVA); ++P < 0.01, compared with negative control cells and STIM1 siRNA-transfected cells (ANOVA). E: siRNA knockdown of STIM1 reduced the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μM CPA in the presence of 10 μM nifedipine. siRNA knockdown of STIM1 and Orai1 further reduced the increase in Mn2+ quench of fura-2 fluorescence caused by CPA in the presence of nifedipine. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in negative control cells (filled bar, n = 125), in STIM1 siRNA-transfected cells (shaded bar, n = 151), and in STIM1 siRNA and Orai1 siRNA-transfected cells (open bar, n = 137). **P < 0.01, compared with negative control cells (ANOVA); ++P < 0.01, compared with the negative control cells and STIM1 siRNA-transfected cells (ANOVA).

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Transfection, Negative Control, Expressing, Western Blot, Knockdown, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: knockdown of Orai1 reduced CCE in STIM1-overexpressing mouse PASMCs. A and B: STIM1, Orai1, and GAPDH proteins were detected in cells infected with adenovirus containing green fluorescent protein (Ad-GFP) transfected with 200 nM scrambled siRNA. The expression of STIM1 but not Orai1 or GAPDH increased markedly in cells infected with STIM1-GFP-adenovirus (Ad-GFP-STIM1) transfected with scrambled siRNA. The expression of Orai1 but not STIM1 or GAPDH was reduced significantly in STIM1-overexpressing cells transfected with 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: overexpression of STIM1 in scrambled siRNA-transfected cells caused an increase in CPA-induced transient and sustained rise in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. The increases in fluorescence ratio were reduced in Orai1 siRNA-transfected cells overexpressed with STIM1. D: bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in GFP-infected cells transfected with scrambled siRNA (filled bars, n = 33), in STIM1-overexpressing cells transfected with scrambled siRNA (shaded bars, n = 65), and in STIM1-overexpressing cells transfected with Orai1 siRNA (open bars, n = 50). *P < 0.05, compared with GFP-infected cells transfected with scrambled siRNA and STIM1-overexpressing cells transfected with Orai1 siRNA (ANOVA). E: overexpression of STIM1 in scrambled siRNA-transfected cells caused a CPA-induced increase in Mn2+ quench of fura-2 fluorescence in the presence of 10 μM nifedipine. The increases in Mn2+ quench of fura-2 fluorescence was reduced in Orai1 siRNA-transfected cells overexpressed with STIM1. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in GFP-infected cells transfected with scrambled siRNA (filled bars, n = 40), in STIM1-overexpressing cells transfected with scrambled siRNA (shaded bars, n = 95), and in STIM1-overexpressing cells transfected with Orai1 siRNA (open bars, n = 57). **P < 0.01, compared with GFP-infected cells transfected with scrambled siRNA and STIM1-overexpressing cells transfected with Orai1 siRNA (ANOVA).

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Knockdown, Infection, Transfection, Expressing, Western Blot, Over Expression, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 coimmunoprecipitates with STIM1 in mouse PASMCs. A, left: Orai1 was detected in cultured mouse PASMCs in the absence and presence of store-depletion. Right: bar graph showing expression levels of Orai1 measured relative to GAPDH in control cells (denoted as 1, filled bar) and in cells subjected to store-depletion (open bar). Data are means ± SE of 7 separate Western blot analyses. B, left: STIM1 was detected in cultured mouse PASMCs in the absence and presence of store-depletion. Right: bar graph showing expression levels of STIM1 measured relative to GAPDH in control cells (denoted as 1, filled bar) and in cells subjected to store-depletion (open bar). Data are means ± SE of 7 separate Western blot analyses. C: STIM1 coimmunoprecipitated Orai1 in cultured mouse PASMCs in the absence and presence of store-depletion. STIM1 was first immunoprecipitated (IP) with EXBIO STIM1 antibody (10 μg), and the blot was subsequently probed with BD Biosciences STIM1 antibody (WB, 1:100). The blot was then probed for coimmunoprecipitation (co-IP) of Orai1 expression using Orai1 antibody (WB, 1:100, ProSci). Experiments were performed in 3 separate co-IP procedures and Western blot analyses.

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Cell Culture, Expressing, Control, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Colocalization of Orai1 and STIM1 in mouse PASMCs. A–D: staining of mouse cultured PASMCs after exposure of live cells with normal bath physiological salt solution (PSS). A: omission of Orai1 and STIM1 antibody resulted in no Orai1 or STIM1 staining. B–D: three representative cells dual-labeled with anti-Orai1 antibody (green) and STIM1 antibody (red). Orai1 and STIM1 colocalization (yellow/orange) is shown in the merged images. E–H: staining of mouse cultured PASMCs after exposure of live cells with Ca2+-free PSS containing 10 μM CPA. E: omission of Orai1 and STIM1 antibody resulted in no Orai1 or STIM1 staining. F–H: three representative cells dual-labeled with anti-Orai1 antibody (green) and STIM1 antibody (red). Colocalization of Orai1 and STIM1 is more apparent (yellow/orange) after store-depletion as shown in the merged images. Nuclei were stained with DAPI (blue). Experiments were performed in 3 separate immunostaining procedure, each with duplicate coverslips. Scale bars, 20 μm.

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Staining, Cell Culture, Labeling, Immunostaining

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 2. Inhibition of junctional communication by Cx43 siRNA and pharmacological inhibitor TPA. (A) Western blotting probed with anti-Cx43 antibody shows the decrease of the Cx43 level in siRNA-transfected cells. (B and C) Cx43-siRNA and TPA reduce dye coupling through gap junction as measured by parachute dye-coupling assay. Columns, mean for five experiments; bars, SEM. **, Significantly different from control, P<0.01.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Inhibition, Western Blot, Transfection, Control

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 3. Simvastatin attenuates cisplatin toxicity at high cell density and this effect was blocked by Cx43-siRNA. (A) Clonogenic survival of cells pretreated with 10 µM simvastatin for 3 h, and co-incubated with 5 µM cisplatin for 1 h at high and low cell density. (B) Clonogenic survival of cells pretreated with a range of concentrations of simvastatin for the indicated periods, followed by co-incubation with 5 µM cisplatin for 1 h at high cell density. (C) Clonogenic survival of siRNA-transfected cells pretreated with 10 µM simvastatin for 3 h, and co-incubated with 5 µM cisplatin for 1 h at high and low cell density. Columns, mean for five experiments; bars, SEM. *, Significantly different from control, P<0.05; #, significantly different from the cisplatin bar, P<0.05.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Incubation, Transfection, Control

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 4. Effects of simvastatin on gap junction intercellular communication, Cx43 immunostaining. (A) The dye spread of cells exposed to increasing concentrations of simvastatin for 4 h as assayed by parachute dye-coupling assay. (B) Immunostaining for Cx43 in cells with or without 10 µM simvastatin treatment. Magnification, x400.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Immunostaining

Journal: Oncology reports

Article Title: Simvastatin protects Sertoli cells against cisplatin cytotoxicity through enhanced gap junction intercellular communication.

doi: 10.3892/or.2015.4192

Figure Lengend Snippet: Figure 5. Effects of simvastatin on Cx43 expression and p-Cx43 (ser368) expression. (A) Western blot analysis of Cx43 expression in cells treated with varying concentrations of simvastatin. (B) Western blotting showing p-Cx43 (ser368) levels in cells following treatment with 10 µM simvastatin for varying time periods. Bar graphs are derived from the densitometric scanning of the blots and shows a comparison of the level of Cx to β-tubulin density ratio. Columns, mean from four experiments; bars, SEM. *, Significantly different from control.

Article Snippet: Individual membranes were probed with monoclonal antiCx43 antibody produced in mouse (1:4,000; Sigma-Aldrich) and monoclonal antibodies against p-Cx43 (ser368) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Western Blot, Derivative Assay, Comparison, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Connexin37‐Dependent Mechanisms Selectively Contribute to Modulate Angiotensin II ‐Mediated Hypertension

doi: 10.1161/JAHA.118.010823

Figure Lengend Snippet: Loss of Cx37 alters the basal expression of aortic Cx43, and protects against the overexpression of Cx40 and Cx45 after the 2K1C procedure. A , Four weeks after the 2K1C procedure, Western blots showed a marked increase of Cx37 levels in the aortas of the WT mice that had become hypertensive. WT sham N=9; Cx37−/− sham N=7; WT 2K1C N=9; Cx37−/− 2K1C N=11. B , The levels of Cx40 were also increased in the aortas of WT made hypertensive, but not in Cx37−/− mice. WT sham N=18; Cx37−/− sham N=15; WT 2K1C N=16; Cx37−/− 2K1C N=14. C , The levels of Cx43 were enhanced in response to a renin‐dependent model of hypertension ( WT 2K1C). Cx37‐deficient mice also displayed elevated Cx43 protein levels, which decreased after the 2K1C procedure. WT sham N=17; Cx37−/− sham N=13; WT 2K1C N=14; Cx37−/− 2K1C N=17. D , Comparable levels of Cx45 were evaluated in the aortas of control WT and Cx37−/− mice. After the 2K1C surgery, these levels did not change in the aortas of WT mice, but decreased in those of Cx37−/− mice. WT sham N=9; Cx37−/− sham N=7; WT 2K1C N=9; Cx37−/− 2K1C N=11. Results are means+ SEM . * P ≤0.05, ** P ≤0.01 vs respective control mice; °° P ≤0.01, °°° P ≤0.001 vs WT mice, as given by 2‐way ANOVA . E , After the 2K1C surgery, the immunostaining of Cx40 (upper panels) was increased between the aortic EC of WT , but not of Cx37−/−mice. Analogous changes were seen with regard to the immunostaining of Cx43 (middle panels) between the SMC of the media. The immunostaining of Cx37 (lower panels) increased in the aortic media of WT mice after the 2K1C surgery. L indicates lumen; M, media; Bar=20 μm. 2K1C indicates 2‐kidney, 1‐clip; Cx37, Connexin37; Cx40, Connexin40; Cx43, Connexin43; Cx45, Connexin45; EC , endothelial cells; Sham, sham‐operated controls; SMC , smooth muscle cells; WT , wild type.

Article Snippet: The membranes were then incubated overnight at 4°C with 1 of the following primary antibodies: rabbit polyclonal antibodies against AT1R (Alomone labs, AAR‐011, 1:1000), AT2R (Alomone labs, AAR‐012, 1:1000), MLC2 (Cell Signaling, 3672, 1:1000), P‐MLC2 (Cell Signaling, 3674, 1:1000), ERK (Cell Signaling, 4695, 1:1000), P‐ERK (Cell Signaling, 9101, 1:1000), AKT (Cell Signaling, 9272S, 1:1000), Cx40 (Chemicon, AB1726; 1:250), Cx37 (Biotrend Chemikalien, Cx37A11‐A; 1:500), Cx43 (Cell Signaling, 3512S, 1:500) or Cx45 (Millipore, AB1745, 1:500); mouse monoclonal antibodies against eNOS (BD Biosciences, 610297, 1:500), PeNOS (BD Biosciences, 612392, 1:500), P‐AKT (Cell Signaling, 4051, 1:500), and α‐tubulin (Sigma‐Aldrich, T5168; 1:2500).

Techniques: Expressing, Over Expression, Western Blot, Control, Mouse Assay, Immunostaining