Journal: Journal of cellular and molecular medicine

Article Title: Termination and activation of store-operated cyclic AMP production.

doi: 10.1111/j.1582-4934.2012.01592.x

Figure Lengend Snippet: Fig. 3 Effect of knockdown of AC isoforms. (A) Response to ionomycin in cells expressing shRNA against AC3; dsRed was used as a marker for transfection with shRNA. (B) Summary of experiments in cells expressing dsRed and shRNA against AC3 (13 exp./16 cells), AC5 (Δ: 7 exp., 12 cells; slope: 6 exp./11 cells), and treated with scramble (Δ: 10 exp./16 cells; slope: 9 exp./15 cells). Amplitude and slope of ionomycin response are expressed as % of control response. (C) Response to ionomycin in cells treated with siRNA for AC3 compared with cells treated with scramble (average of 14 scramble and 12 dsRed cells) (D) Summary of experiments with cells treated with siRNA for AC3 (6 exp./80 cells), AC5 (7 exp./68 cells) and AC6 (8 exp./91 cells) expressed as % of the response in scramble cells (scramble controls for siAC3 and siAC5: 13 exp./126 cells; scram- ble controls for siAC6 16 exp./202 cells). (E) Summary of qPCR experiments. siRNA AC3: 3 independent experimental samples; siRNA AC5: 3 exp.; siRNA AC6: 2 exp. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Validation of three different strategies used for STIM2 silencing: (i) Dharmacon SmartPool (siRNA STIM2-a), (ii) Ambion silencer (siRNA STIM2-b), and (iii) Origene shRNA (shRNA STIM2).

Techniques: Knockdown, Expressing, shRNA, Marker, Transfection, Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Differential expression of ORAI channels and STIM proteins in renal cell carcinoma subtypes: implications for metastasis and therapeutic targeting

doi: 10.4196/kjpp.24.126

Figure Lengend Snippet: (A, C) FPKM for Orai3 according to normal and RCC grades (Stage I, II, III, and IV) in clear cell RCC (ccRCC) and papillary RCC (pRCC), respectively. Box whisker plots express the minimum, median, and maximum values of FPKM (expressed as log(expr + 1)), where FPKM was adjusted using a scaling factor instead of total mapped reads to remove a million units. ***p ≤ 0.001 of one-way ANOVA (normal vs . tumor stages). (B, D) Kaplan–Meler survival analysis comparing females (left) and males (right) in the low and high mRNA levels of Orai3 in ccRCC and pRCC, respectively. The cut-off value for Orai3 in ccRCC is 13.5, and in pRCC is 16.87. (E, G) Correlation analysis between gene copy number (log2-transformed values) of STIM2 and Orai3 in ccRCC (total n = 355) and pRCC (total n = 241), respectively. The linear regression line with a 95% confidence interval and the Spearman r correlation coefficient. (F, H) Kaplan–Meler survival analysis comparing males and females with high Orai3 and low STIM2 mRNA levels in ccRCC and pRCC, respectively. The cut-off value for Orai3 is 13, and for STIM2 is 4.5. p-values were determined by a log-rank test (B, D, F, and H).

Article Snippet: The primary antibodies used for immunoblotting were: β-actin (1:3,000, #sc-47778) and Cyclin D1 (1:200, #sc-246) from Santa Cruz Biotechnology; STIM1 (1:1,000, #S6072) from Sigma Aldrich; p-FAK (Tyr397) (1:1,000, #8556) and t-FAK (1:1,000, #13009) from Cell Signaling Technology Inc.; and STIM2 (1:500, #21192-1-AP) from ProteinTech.

Techniques: Whisker Assay, Transformation Assay

Journal: Frontiers in Physiology

Article Title: Store-operated calcium entry and the localization of STIM1 and Orai1 proteins in isolated mouse sinoatrial node cells

doi: 10.3389/fphys.2015.00069

Figure Lengend Snippet: List of antibody used in the study .

Article Snippet: STIM2 , ProSci (4123) , Polyclone , Rabbit , 1:400 , 1:1000.

Techniques: Staining, Western Blot, Transduction

Journal: Frontiers in Physiology

Article Title: Store-operated calcium entry and the localization of STIM1 and Orai1 proteins in isolated mouse sinoatrial node cells

doi: 10.3389/fphys.2015.00069

Figure Lengend Snippet: Expression of STIM and Orai molecules in mouse cardiac myocytes. (A) Total RNA was isolated from pacemaker cells (SAN), atrial myocytes (Atrium), ventricular myocytes (Ventricle) and spleen. Transcripts of Stim1, Stim2, Orai1, Orai2, and Orai3 in cardiac myocytes were compared with the spleen as positive control. The housekeeping gene Hypoxanthine–Guanine Phosphoribosyltransferase (HPRT1) was used as an internal control. (B) Protein levels of STIM1, STIM2, Orai1, and Orai3 were examined in total protein extracts from SAN region (SAN), atrium (Atrium), ventricle (Ventricle) and spleen and determined by Western Blot analysis. The nuclear protein histone was used as loading control. (C) Bar graph showing quantitative western blot analysis of STIM1 protein levels in spleen and heart with heart samples normalized to spleen as 1 ( n = 3 gels, P < 0.05, ANOVA). (D) Confocal images of an isolated pacemaker cell shows expression of HCN4 (green in color), a positive marker for pacemaker cells. The cell also positively stained with anti-Orai1 antibody (red in color). The nucleus is stained blue with DAPI. A merged image on the left shows the co-localization of HCN4 and Orai1 along the surface membrane (color in yellow). (E) Representative confocal images of an isolated pacemaker cell labeled with STIM1 (green in color) and DAPI (blue in color). When perfused with normal Ca 2+ containing solution, positive STIM1 staining was distributed both intracellularly and in the cell periphery as showed in the top panel. When perfused with Ca 2+ -free solution containing thapsigargin, STIM1 peripheral localization was enhanced, as shown in the bottom panel.

Article Snippet: STIM2 , ProSci (4123) , Polyclone , Rabbit , 1:400 , 1:1000.

Techniques: Expressing, Isolation, Positive Control, Control, Western Blot, Marker, Staining, Membrane, Labeling

Journal: Biochimica et biophysica acta

Article Title: Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

doi: 10.1016/j.bbamcr.2008.11.008

Figure Lengend Snippet: Fig. 2. Cellular level of STIM1 protein is elevated and that of STIM2 is decreased in MEF DKO cells. (A,D) Representative immunoblots of protein extracts from B cell lines developed with anti-STIM1 and anti-STIM2 antibodies and results of densitometric analysis of five independent experiments. (B,C) Results of RT-PCR quantification of STIM1 and STIM2 mRNA levels in MEF cell lines derived from wt, PS1−/−, PS2−/−or DKO (lacking both PS1 and PS2) animals.

Article Snippet: As a double band was seen on immunoblots on human B lymphocytes and HEK293 cells developed with anti-STIM2 antibody (ProSci Incorporated), we analyzed the lower band which, according to antibody manufacturer information, represents STIM2 protein.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Biochimica et biophysica acta

Article Title: Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

doi: 10.1016/j.bbamcr.2008.11.008

Figure Lengend Snippet: Fig. 3. CCE is attenuated but STIM proteins' levels are not affected by overexpression of PS1 FAD mutants in HEK293 cells. (A) Averaged traces obtained by ratiometric Fura-2 analysis of HEK293 cell lines overexpressing wt PS1 and PS1 P117R and PS1 E318G mutants. (B) Results of quantification of CCE in analyzed HEK293 cell lines (⁎⁎⁎Pb0,001). (C) Representative immunoblot of protein extracts from HEK293 cell lines developed with anti-STIM1 and anti-STIM2 (D) antibodies and results of densitometric analysis of three independent experiments.

Article Snippet: As a double band was seen on immunoblots on human B lymphocytes and HEK293 cells developed with anti-STIM2 antibody (ProSci Incorporated), we analyzed the lower band which, according to antibody manufacturer information, represents STIM2 protein.

Techniques: Over Expression, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

doi: 10.1016/j.bbamcr.2008.11.008

Figure Lengend Snippet: Fig. 5. STIM2 mRNA and protein levels are decreased in B lymphocytes isolated from FAD patients. (A,C) Results of RT-PCR quantification of STIM1 and STIM2 mRNA levels in cell populations from healthy controls and FAD patients. (B,D) Representative immunoblot of protein extracts from B lymphocytes lines developed with anti-STIM1 and anti-STIM2 (D) antibodies and results of densitometric analysis of three independent experiments.

Article Snippet: As a double band was seen on immunoblots on human B lymphocytes and HEK293 cells developed with anti-STIM2 antibody (ProSci Incorporated), we analyzed the lower band which, according to antibody manufacturer information, represents STIM2 protein.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Hypertension

Article Title: STIM2 (Stromal Interaction Molecule 2)–Mediated Increase in Resting Cytosolic Free Ca 2+ Concentration Stimulates PASMC Proliferation in Pulmonary Arterial Hypertension

doi: 10.1161/hypertensionaha.117.10503

Figure Lengend Snippet: Figure 1. STIM2 (stromal interaction molecule 2) protein expression is upregulated in pulmonary artery smooth muscle cell (PASMC) from patients with pulmonary arterial hypertension (PAH) compared with PASMC from normal subjects and is sufficient and necessary for increasing the resting [Ca2+]cyt in PASMCs. A, Representative fluorescent images showing the resting [Ca2+]cyt in a normal PASMC and a PAH PASMC. B, Histogram (left) showing the distribution of the resting [Ca2+]cyt in normal (n=3 normal subjects) and PAH (n=5 patients) PASMCs. Summarized data (right, means±SE) showing the averaged resting [Ca2+]cyt in normal and PAH PASMCs. ***P<0.001 vs normal. C, Western blot analyses on STIM2 (left) in normal and PAH PASMCs. Summarized data (right, means±SE, n=3 normal subjects, and 4 patients with PAH) showing the protein level of STIM2 in normal and PAH PASMCs. *P<0.05 vs normal. D, Representative fluorescent images (a) showing the resting [Ca2+]cyt in normal PASMCs and normal PASMCs transfected with GFP- vector and STIM2 gene (STIM2-YFP [yellow fluorescent protein]). Arrows indicate the normal PASMCs that are positively transfected with STIM2-YFP. Histogram (b) showing the distribution of the resting [Ca2+]cyt in control, GFP-vector–transfected and STIM2- YFP–transfected normal PASMCs. Vertical arrows indicate the peak values of the resting [Ca2+]cyt. Summarized data (c; means±SE, n=3 normal subjects) showing the averaged resting [Ca2+]cyt in control, GFP-vector–transfected and STIM2-YFP–transfected normal PASMCs. ***P<0.001 vs control and GFP-vector. E, Representative fluorescent images (a) showing the resting [Ca2+]cyt in PAH-PASMCs (control) and PAH-PASMCs transfected with a control-CRISPR/Cas9 plasmid (control-CRISPR/Cas9) and the STIM2-CRISPR/Cas9 plasmid (STIM2-CRISPR/Cas9). Arrows indicate the PAH-PASMCs that are positively transfected with STIM2-CRISPR/Cas9 plasmid. Histogram (b) showing the distribution of the resting [Ca2+]cyt in control, control-CRISPR/Cas9–transfected and STIM2-CRISPR/Cas9– transfected PAH-PASMCs, with the averaged values of the resting [Ca2+]cyt at 138.2, 132.5, and 91.8 nmol/L, respectively. Summarized data (c; means±SE, n=5 patients with PAH) showing the level of the resting [Ca2+]cyt in control, control-CRISPR/Cas9–transfected and STIM2-CRISPR/Cas9–transfected PAH-PASMC. ***P<0.001 vs control and control-CRISPR/Cas9.

Article Snippet: CRISPR/Cas9 and small interfering RNA 110 used for downregulation of STIM2 include: STIM2 CRISPR/Cas9 (Santa Cruz Biotechnology, 111 Cat.# sc-403491), STIM2-siRNA (Ambion Life Technology, Cat.# 4392420) and control-112 siRNA (Santa Cruz Biotechnology, Cat.# sc-37007).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control, CRISPR

Journal: Hypertension

Article Title: STIM2 (Stromal Interaction Molecule 2)–Mediated Increase in Resting Cytosolic Free Ca 2+ Concentration Stimulates PASMC Proliferation in Pulmonary Arterial Hypertension

doi: 10.1161/hypertensionaha.117.10503

Figure Lengend Snippet: Figure 2. STIM2 (stromal interaction molecule 2) is necessary for increasing the level of phosphorylated (p) CREB (cAMP response element–binding protein), STAT3 (signal transducer and activator of transcription 3), and AKT and enhancing the nuclear translocation of NFATC2 (nuclear factor of activated T-cells, cytoplasmic 2) in pulmonary artery smooth muscle cells (PASMCs). A, Western blot analyses (a) on pCREB, CREB, pSTAT3, STAT3, pAKT, and AKT in normal and pulmonary arterial hypertension (PAH) PASMCs. Summarized data (b; means±SE, n=3 normal subjects and 5 patients with PAH) showing the protein level of pCREB, CREB, pSTAT3, STAT3, pAKT, and AKT in normal and PAH PASMC. *P<0.05 and **P<0.01 vs normal. B, Western blot analyses (a) on pCREB, CREB, pSTAT3, STAT3, pAKT, and AKT in PAH-PASMCs transfected with control-siRNA and STIM2-siRNA. Summarized data (b; means±SE, n=5 patients with PAH) showing the protein level of pCREB, CREB, pSTAT3, STAT3, pAKT, and AKT in control-siRNA– and STIM2-siRNA–transfected PAH- PASMCs. *P<0.05 vs control-siRNA. C, Representative fluorescent images showing NFATC2 (red), STIM2 (green), and nuclei (blue) in control-siRNA–transfected normal and PAH-PASMCs and STIM2-siRNA–transfected PAH-PASMCs. Summarized data (right, means±SE, n=3 normal subjects, and 5 patients with PAH) showing the percentage of NFATC2-activated cells in control-siRNA–transfected normal and PAH-PASMCs and STIM2-siRNA–transfected PAH-PASMCs. **P<0.01 vs nor control-siRNA and PAH STIM2-siRNA.

Article Snippet: CRISPR/Cas9 and small interfering RNA 110 used for downregulation of STIM2 include: STIM2 CRISPR/Cas9 (Santa Cruz Biotechnology, 111 Cat.# sc-403491), STIM2-siRNA (Ambion Life Technology, Cat.# 4392420) and control-112 siRNA (Santa Cruz Biotechnology, Cat.# sc-37007).

Techniques: Binding Assay, Translocation Assay, Western Blot, Transfection, Control

Journal: Hypertension

Article Title: STIM2 (Stromal Interaction Molecule 2)–Mediated Increase in Resting Cytosolic Free Ca 2+ Concentration Stimulates PASMC Proliferation in Pulmonary Arterial Hypertension

doi: 10.1161/hypertensionaha.117.10503

Figure Lengend Snippet: Figure 3. STIM2 (stromal interaction molecule 2) is sufficient for increasing the level of phosphorylated (p) CREB (cAMP response element–binding protein), STAT3 (signal transducer and activator of transcription 3), and AKT and enhancing the nuclear translocation of NFATC2 (nuclear factor of activated T-cells, cytoplasmic 2) in pulmonary artery smooth muscle cells (PASMCs). A, Western blot analyses (a) on pCREB, CREB, pSTAT3, STAT3, pAKT, and AKT in vector- and STIM2-YFP (yellow fluorescent protein)–transfected normal PASMCs. Summarized data (b; means±SE, n=3 normal subjects) showing the protein level of pCREB, CREB, pSTAT3, STAT3, pAKT, and AKT in vector- and STIM2-YFP–transfected normal PASMC. *P<0.05 vs vector. B, Western blot analyses (a) on pCREB, CREB, pSTAT3, STAT3, pAKT, and AKT in vector- and STIM2-YFP–transfected normal PASMCs treated without and with EGTA. Summarized data (b, means±SE, n=3 normal subjects) showing the protein level of pCREB, CREB, pSTAT3, STAT3, pAKT, and AKT in vector- and STIM2-YFP–transfected normal PASMCs treated without and with EGTA. *P<0.05 vs vector and STIM2-YFP+EGTA. C, Representative fluorescent images showing NFATC2 (red), STIM2 (green), and nuclei (blue) in vector- and STIM2-YFP–transfected normal PASMCs treated without and with EGTA. Summarized data (right, means±SE, n=3 normal subjects) showing the percentage of NFATC2-activated cells in vector- and STIM2- YFP–transfected normal PASMCs treated without and with EGTA. ***P<0.001 vs vector and STIM2-YFP+EGTA. D, Summarized data (means±SE, n=3 normal subjects) showing the resting [Ca2+]cyt in vector- and STIM2-YFP–transfected normal PASMCs treated without and with EGTA. ***P<0.001 vs vector and STIM2-YFP+EGTA.

Article Snippet: CRISPR/Cas9 and small interfering RNA 110 used for downregulation of STIM2 include: STIM2 CRISPR/Cas9 (Santa Cruz Biotechnology, 111 Cat.# sc-403491), STIM2-siRNA (Ambion Life Technology, Cat.# 4392420) and control-112 siRNA (Santa Cruz Biotechnology, Cat.# sc-37007).

Techniques: Binding Assay, Translocation Assay, Western Blot, Plasmid Preparation, Transfection

Journal: Hypertension

Article Title: STIM2 (Stromal Interaction Molecule 2)–Mediated Increase in Resting Cytosolic Free Ca 2+ Concentration Stimulates PASMC Proliferation in Pulmonary Arterial Hypertension

doi: 10.1161/hypertensionaha.117.10503

Figure Lengend Snippet: Figure 4. STIM2 (stromal interaction molecule 2) is sufficient and necessary for increasing cell proliferation in pulmonary artery smooth muscle cells (PASMCs). A, Representative fluorescent images (a) showing nuclei (blue) and 5-ethynyl-2-deoxyuridine (EdU; red), along with the overlay images (purple), in vector- and STIM2-transfected normal PASMCs. Summarized data (b; means±SE, n=3) showing the percentage of cells with EdU incorporation (middle) and averaged cell number per field (right) in vector- and STIM2-transfected normal PASMCs. *P<0.05 vs vector. B, Representative fluorescent images (a) showing nuclear (blue) and EdU staining (red), as well as the overlay images (purple), in control-siRNA– and STIM2-siRNA–transfected pulmonary arterial hypertension (PAH)–PASMCs. Summarized data (b; means±SE, n=5) showing the percentage of cells with EdU incorporation (middle) and averaged cell number per field (right) in control-siRNA– and STIM2-siRNA–transfected PAH-PASMCs. *P<0.05 vs control-siRNA. C, Representative fluorescent images showing STIM2 (green), Ki67 (red), and nuclei (blue) in normal PASMCs transfected with vector and STIM2-YFP (yellow fluorescent protein), and PAH-PASMCs transfected with control-siRNA and STIM2-siRNA. Summarized data (left, means±SE, n=3 normal subjects and 5 patients with PAH) showing the percentage of Ki67-positive normal PASMCs (left) transfected with vector and STIM2-YFP, and Ki67-positive PAH- PASMCs (right) transfected with control-siRNA and STIM2-siRNA, respectively. *P<0.05 vs vector and **P<0.01 vs control-siRNA.

Article Snippet: CRISPR/Cas9 and small interfering RNA 110 used for downregulation of STIM2 include: STIM2 CRISPR/Cas9 (Santa Cruz Biotechnology, 111 Cat.# sc-403491), STIM2-siRNA (Ambion Life Technology, Cat.# 4392420) and control-112 siRNA (Santa Cruz Biotechnology, Cat.# sc-37007).

Techniques: Plasmid Preparation, Transfection, Staining, Control

Journal: Hypertension

Article Title: STIM2 (Stromal Interaction Molecule 2)–Mediated Increase in Resting Cytosolic Free Ca 2+ Concentration Stimulates PASMC Proliferation in Pulmonary Arterial Hypertension

doi: 10.1161/hypertensionaha.117.10503

Figure Lengend Snippet: Figure 6. STIM2 (stromal interaction molecule 2) clustering is increased in pulmonary arterial hypertension–pulmonary artery smooth muscle cells (PAH-PASMCs). A, Representative fluorescent images (left) showing the nuclei (blue), STIM2 (green), and Orai1 (red), along with the overlay images (orange), in normal and PAH-PASMCs treated with cyclopiazonic acid (CPA; 10 µmol/L). PASMCs treated with CPA exhibit a STIM2 distribution in the plasma membrane. Overlay images showing the colocalization of STIM2 and Orai1 in the plasma membrane in PASMCs after CPA treatment (indicated by arrows). The magnified images (highlighted in yellow) showing the STIM2 clusters (red) and STIM2/Orai1 interactions in the sarcoplasmic reticulum (SR)-plasma membrane junction area. Line scan measurement (middle) showing different fluorescence intensity (representing STIM2 and STIM2/Orai1 levels) along the line of the region indicated in the left images. Summarized data (right, means±SE, n=3 normal subjects, and 5 patients) showing the fluorescent intensity of the clusters in normal and PAH-PASMCs with CPA treatment. B, Representative fluorescent images (upper) showing the nuclei (blue), STIM2 (green) and Orai1 (red), along with the overlay images (orange), in normal and PAH-PASMCs without CPA treatment. PAH-PASMCs exhibit STIM2 clustering distributions in the plasma membrane. Overlay images showing the colocalization of STIM2 and Orai1 in the plasma membrane (arrows). The magnified images (highlighted in yellow) showing the STIM2 clusters (red) and STIM2/Orai1 interactions in the SR-plasma membrane junction area. Line scan measurement (middle) showing the different fluorescence intensity (representing STIM2 and STIM2/ Orai1 levels) along the line of the region indicated in the left images. Summarized data (lower, right, means±SE, n=3 normal subjects, and 5 patients) showing the cluster intensity in normal and PAH-PASMCs without CPA treatment. C, Western blot analyses on STIM2 in normal and PAH-PASMCs before and after deglycosylation using PNGaseF.

Article Snippet: CRISPR/Cas9 and small interfering RNA 110 used for downregulation of STIM2 include: STIM2 CRISPR/Cas9 (Santa Cruz Biotechnology, 111 Cat.# sc-403491), STIM2-siRNA (Ambion Life Technology, Cat.# 4392420) and control-112 siRNA (Santa Cruz Biotechnology, Cat.# sc-37007).

Techniques: Clinical Proteomics, Membrane, Fluorescence, Western Blot