Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

doi: 10.1159/000495229

Figure Lengend Snippet: Fig. 2. Effect of lithium treatment without and with NFκB inhibitor wogonin on Orai1, STIM1 and STIM2 protein abundance in iPSC-derived neurons from ChAc patients. A,B,C. Original Western blot of (A) Orai1, (B) STIM1 and (C) STIM2 protein abundance in neurons differentiated from iPSCs derived from ChAc patients (ChAc) without or with lithium (24 h, 2 mM) treatment without and with presence of NFκB inhibitor wogonin (50 µM). D,E,F. Arithmetic means (± SEM, n = 5) of (D) Orai1, (E) STIM1 and (F) STIM2 protein abundance in neurons generated from iPSCs derived from ChAc patients without (blue bars) or with (red and grey bars) lithium (24 h, 2 mM) treatment without (red bars) and with (grey bars) presence of NFκB inhibitor wogonin (50 µM). #(p<0.05), ##(p<0.01) indicates statistically significant difference to respective value in the absence of treatment, §(p<0.05), §§§(p<0.001) indicates statistically significant difference to respective value in the absence of wogonin.

Article Snippet: The proteins were separated by 10% SDS-PAGE in Glycine-Tris buffer and electro-transferred onto nitrocellulose membranes for 90 min. After blocking with 5% milk in TBST at room temperature for 1 h, the membranes were incubated with primary anti-ORAI1 antibody (1:1000, Proteintech), anti-STIM1 antibody (1:1000, Cell Signaling), anti-STIM2 antibody (1:1000, Cell Signaling) and anti-GAPDH antibody (1:1000, Cell Signaling) at 4°C overnight.

Techniques: Quantitative Proteomics, Derivative Assay, Western Blot, Generated

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Co-localization and co-immunoprecipitation of TRPP2 and STIM1 in transfected HEK293 cells. a Upper penal: GFP-tagged TRPP2 (TRPP2-GFP) co-localized with endoplasmic reticulum maker (ER-DsRed); Lower penal: TRPP2-GFP co-localized with mCherry-tagged STIM1 (mCherry-STIM1). b Representative images showing co-immunoprecipitation followed by immunoblots [left, immunoblot with anti-GFP; right, immunoblot with anti-mCherry]. GFP or mCherry antibody pulled down the proteins from TRPP2-GFP and mCherry-STIM1 co-expressing HEK293 cells. The experiment was repeated 4 times

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Immunoprecipitation, Transfection, Western Blot, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: FRET efficiency of TRPP2-STIM1 interaction in TRPP2 and STIM1 co-expressing HEK293 cells. a Representative images showing GFP, mCherry and FRET efficiency channels. The cells expressed TRPP2-mCherry and STIM1-GFP (upper), mCherry-TRPP2 and STIM1-GFP (middle) or GFP-mCherry (lower) proteins respectively. b Summarized data showing FRET efficiency in transfected HEK293 cells (four groups: mCherry and GFP, mCherry-GFP, TRPP2-mCherry and STIM1-GFP, mCherry-TRPP2 and STIM1-GFP). Values are shown as the mean ± SEM ( n = 9–20 cells). * P < 0.05 for GFP and mCherry co-expression vs. GFP-mCherry expression; # P < 0.05 for TRPP2-mCherry and STIM1-GFP co-expression vs. mCherry-TRPP2 and STIM1-GFP co-expression. c Schematic diagram of full-length TRPP2 and its truncated derivatives: TRPP2 without N1 (2-111aa, ∆N1) and TRPP2 without N2 (112-221aa, ∆N2). d Representative images of co-immunoprecipitation experiments in HEK293 cells co-expressed with STIM1 plus GFP-tagged full-length TRPP2 (WT) or ∆N1 or ∆N2. IP, GFP antibody; IB, anti-EGFP antibody. The GFP-only vector was used as a negative control. e Summarized data showing the relative binding strength of STIM1 with TRPP2 (WT), or ∆N1 or ∆N2. The optical density of ∆N1 or ∆N2 blot was normalized to that of WT blot (= 100%) and expressed as the relative binding strength. Values are shown as the mean ± SEM ( n = 3). * P < 0.05 for STIM1 and ∆N1 or ∆N2 co-expression vs. STIM1 and TRPP2 co-expression

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Expressing, Transfection, Immunoprecipitation, Plasmid Preparation, Negative Control, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Effect of TRPP2-STIM1 interaction on store-operated Ca 2+ entry (SOCE) in TRPP2 and STIM1 co-expressing HEK293 cells. a - d Summary of data showing changes in Ca 2+ release ( a , c ) and SOCE ( b , d ) in HEK293 cells transfected with TRPP2 siRNA, STIM1 siRNA, STIM1, TRPP2 and/or dominant negative TRPP2 (D511V), and treated by ATP (10 μmol/L), 2APB (100 μmol/L) + ATP (10 μmol/L) and thapsigargin (TG, 2.5 μmol/L) for 8 min in Ca 2+ -free solution. SOCE was evoked by extracellular Ca 2+ (1 mmol/L) application. Values are shown as the mean ± SEM ( n = 3–5). * P < 0.05 compared with scrambled siRNA in each treatment. e Summarized data showing ATP (10 μmol/L)-induced SOCE in HEK293 cells co-expressed with STIM1 and GFP-tagged full-length TRPP2 (GFP-TRPP2) or TRPP2 without N1 (GFP-TRPP2-∆N1, deletion of 2-111aa in TRPP2) or TRPP2 without N2 (GFP-TRPP2-∆N2, deletion of 112-221aa in TRPP2). Values are shown as mean ± SEM ( n = 5). * P < 0.05 for STIM1 and GFP-TRPP2 or GFP-TRPP2-∆N2 co-expression vs. STIM1 and GFP-TRPP2-∆N1 co-expression

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Expressing, Transfection, Dominant Negative Mutation

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Co-immunoprecipitation and in situ proximity ligation assay (PLA) of TRPP2 and STIM1 in mouse aortic smooth muscle cells. ( a , b ) Immunoblots showing that anti-TRPP2 and anti-STIM1 recognized TRPP2 and STIM1 proteins, and co-immunoprecipitation followed by immunoblots ( a , immunoblot with anti-TRPP2; b , immunoblot with anti-STIM1). Proteins from the mouse aortic smooth muscle cells were immunoprecipitated with indicated antibody (+) or no antibody (−). ( c ) PLA analysis was used to detect the interaction between TRPP2 and STIM1. Representative images were displayed in the presence of anti-STIM1 antibody alone ((a)-(c)), or in the presence of anti-TRPP2 and anti-STIM1 antibodies ((d)-(f)). ((c), (f)) were merged with bright view. Red doted fluorescence showing positive signal. Nuclei were marked by DAPI staining (blue color). Scale bar represents 5 μm. The experiment was repeated 4 times

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Immunoprecipitation, In Situ, Proximity Ligation Assay, Western Blot, Fluorescence, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Role of TRPP2 in store-operated Ca 2+ entry (SOCE) and STIM1 puncta formation in mouse aortic smooth muscle cells. a Representative traces showing ATP (10 μmol/L)-induced Ca 2+ release in Ca 2+ -free solution and SOCE in the mouse aortic smooth muscle cells transfected with TRPP2, STIM1, both TRPP2 and STIM1, or scrambled siRNAs. b Summary of data showing changes in intracellular Ca 2+ concentration increase in response to extracellular ATP (10 μmol/L) or thapsigargin (TG, 2.5 μmol/L) application in the mouse aortic smooth muscle cells treated with or without 2APB (100 μmol/L) for 10 min in Ca 2+ -free solution. c Summary of data showing changes in intracellular Ca 2+ concentration increase in response to extracellular Ca 2+ (1 mmol/L) application in the mouse aortic smooth muscle cells treated by ATP (10 μmol/L), 2APB (100 μmol/L) + ATP (10 μmol/L) and thapsigargin (TG, 2.5 μmol/L) for 10 min in Ca 2+ -free solution. Values are shown as mean ± SEM ( n = 3–6 experiments). * P < 0.05 for scrambled siRNA vs. TRPP2 or STIM1 or TRPP2 + STIM1 siRNA transfection in each treatment. d Representative images showing STIM1 puncta formation in the mouse aortic smooth muscle cells transfected with TRPP2 siRNA or scrambled siRNA and treated by ATP (10 μmol/L), 2APB (100 μmol/L) + ATP (10 μmol/L) and thapsigargin (TG, 2.5 μmol/L) for 10 min in Ca 2+ -free solution. Scale bar represents 10 μm. The experiment was repeated 4 times

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Transfection, Concentration Assay

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Role of TRPP2 and STIM1 in Ca 2+ release and store-operated Ca 2+ entry (SOCE)-induced mouse aorta contraction. a Representative traces showing phenylephrine (Phe, 10 μmol/L)-induced contraction in Ca 2+ -free solution and extracellular Ca 2+ (2.5 mmol/L) re-addition-induced contraction in mice aortae. b - e Summarized data showing Phe-induced contraction in Ca 2+ -free solution ( b , d ) and extracellular Ca 2+ re-addition ( c , e )-induced contractions in mice aortae, which were transfected with TRPP2 siRNA ( b , c ), SITM1 siRNA ( d , e ) or scrambled siRNA. f - i Summarized data showing endothelin 1 (ET-1, 100 nmom/L)-induced contraction in Ca 2+ -free solution ( f , h ) and extracellular Ca 2+ re-addition ( g , i )-induced contractions in mice aortae transfected with TRPP2 siRNA ( f , g ), SITM1 siRNA ( h , i ) or scrambled siRNA. Values are shown as mean ± SEM ( n = 3–4 mice). * P < 0.05 for scrambled siRNA vs. TRPP2 siRNA or STIM1 siRNA transfection. j Representative traces showing thapsigargin (TG, 2.5 μmol/L)-induced contraction in Ca 2+ -free solution and extracellular Ca 2+ (2.5 mmol/L) re-addition-induced contraction in mice aortae. k - n Summarized data showing TG-induced contraction in Ca 2+ -free solution ( k , m ) and extracellular Ca 2+ re-addition ( l , n )-induced contraction in mice aortae, which were transfected with TRPP2 siRNA ( j - l ), SITM1 siRNA ( m - n ) or scrambled siRNAs. Values are shown as the mean ± SEM ( n = 3–4 mice). * P < 0.05 for TRPP2 siRNA or STIM1 siRNA vs. scrambled siRNA

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: TRPP2 and STIM1 form a microdomain to regulate store-operated Ca 2+ entry and blood vessel tone

doi: 10.1186/s12964-020-00560-7

Figure Lengend Snippet: Role of TRPP2 and STIM1 in agonist-induced mouse aorta contraction. Phenylephrine (10 μmol/L, a , b , e , f ) and endothelin 1 (100 nmol/L, c , d , g , h ) concentration-dependently induced the contraction of the mice aortae transfected with TRPP2 ( a - d ), STIM1 ( e - h ) or scrambled siRNA. b , d , f , h The mice aortae were pretreated by heparin (1 mg/ml) using a reversible permeabilization loading procedure. Values are shown as mean ± SEM ( n = 4–6 mice). * P < 0.05 for scrambled siRNA vs. TRPP2 or STIM1 siRNA transfection

Article Snippet: The primary rabbit antibody against STIM1 was obtained from ProSci.

Techniques: Concentration Assay, Transfection

Journal: Frontiers in Pharmacology

Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: αPD-1 treatment increases K + channel activity in HNSCC T cells. (A) Representative current traces of KCa3.1 and Kv1.3 channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs cells from a HNSCC patient in absence or presence of αPD-1 (10 μg/ml, for 6 h). Data are normalized to maximum current at +50 mV recorded using a ramp pulse protocol from −120 mV to +50 mV for 200 ms every 15 s. The holding potential used was −70 mV. (B,C) KCa3.1 (B) and Kv1.3 (C) conductance (G) measured in the absence or presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 68 cells without pembrolizumab and n = 55 cells with pembrolizumab from 14 patients). (D) Representative current traces of divalent free current (DVF) through CRAC channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs from a HNSCC patient. Data were recorded using a ramp pulse protocol from −100 to +100 mV with at holding potential of +30 mV every 1.5 s. Cells were perfused with 0 mM Ca 2+ solution (1 min) followed by 20 mM Ca 2+ (1 min) and DVF solutions (2 min, see methods) to amplify currents during recordings. (E) Peak DVF current values measured in absence and presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 34 cells without αPD-1 and n = 31 cells with αPD-1 from 8 patients). The values in panels (B,C) and (E) are represented as box plots: the horizontal line indicates the median; the lower box is the 25 th percentile; the upper box is the 75 th percentile; and the whiskers represent the 10 th and 90 th percentiles. (F) Ion channel expression (KCa3.1, Kv1.3, Orai1 and STIM1) in HNSCC patient T cells after treatment with αPD-1 (10 μg/ml for 6 h). Effect of αPD-1 treatment is shown as ratio of mean fluorescence intensity (MFI, fold change) values of treatment versus control group. Data are represented as scatter plot where each symbol represents an individual patient ( n = 4–5). Horizontal line represents mean values for each group. Data in panels (B,C,E) were analyzed by Mann-Whitney rank sum test.

Article Snippet: Cells were then stained for rabbit anti-human STIM1 (Proteintech) primary antibodies followed by secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher).

Techniques: Activity Assay, Incubation, Expressing, Fluorescence, Control, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.

Article Snippet: Cells were then stained for rabbit anti-human STIM1 (Proteintech) primary antibodies followed by secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher).

Techniques: Inhibition, Patch Clamp, Transferring, Control, Fluorescence, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2016.00114

Figure Lengend Snippet: Phemindole downregulates STIM1 Expression. (A) Immunofluorescence images showing decreased expression of STIM1 in the MDAMB-231 cells in response to Phemindole treatment. Anti STIM1 antibody was FITC tagged (green) and nuclei were stained with DAPI (blue) (left panel). Corrected Fluoroscence intensity of STIM1 protein was quantified and represented as bar diagram (Right panel). Pictures showed representative cells of each population and were representative of three independent experiments. (B) Western blot analysis depicting the changes in the expression pattern of STIM1 and Orai1. MDAMB-231 cells were treated with Phemindole and Western blotted with respective antibodies as labeled in the figure (upper-left panel). Quantification of WB were depicted as bar diagram (upper-right panel). MDAMB-231 cells were treated with 10 μM Phemindole for 24 h. Lysates were subjected to SDS–PAGE followed by Western blotting (with anti-Orai1 antibodies. β–actin was used as a loading control (middle-left panel). Band intensity was quantified and represented as a bar histogram (middle-right panel). Cells were treated with 10 μM Phemindole and were harvested at 0, 6, 12, or 24 h after Phemindole treatment. Western blotting was done. Antibodies used are labeled and β-actin was used as loading control (lower-left panel). Lower-right panel represent the normalized intensity of the bands. Blots are representative of three independent experiments and quantifications are indicated by Bar diagram. Values were normalized by intensity of loading control. (C) MDAMB-231 cells were treated with Phemindole or Tg under different conditions, lysed and proteins were subjected to immune precipitation by anti STIM1 antibody and followed by Western blotting with respective antibody (upper panel). (D) Representative Fluorescence images showed the association between STIM1 (red) and ORAI1 (green) at plasma membrane. Nuclei were stained with DAPI (blue). Enlargements of the areas indicated by the color rectangles showed overlap formation of STIM1 and Orai1 in plasma membrane due to Tg treatment (Left-middle) which is invisible in Phemindole treated set (left-lower) and not prominent in un-stimulated control set (left-upper panel). Pictures showed representative cells of each population and were representative of three independent experiments. Right panel, the Pearson’s and Manders coefficients determining the level of overlap of RITC and FITC on treatment with Control, thapsigargin ( TG ) and Phemindole has been represented graphically. ∗ , ∗∗ , ∗∗∗ , # indicate p < 0.05, p < 0.01, p < 0.0005, p > 0.05 respectively versus untreated or control group.)

Article Snippet: MDAMB-231 cells were transfected with 300 pmol of STIM1 siRNA (Santa Cruz Biotechnology) and Lipofectamine 2000 (Invitrogen) separately for 12 h. Levels of STIM1 proteins were estimated by Western blotting.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Labeling, SDS Page, Control, Fluorescence, Clinical Proteomics, Membrane

Journal: Frontiers in Pharmacology

Article Title: Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2016.00114

Figure Lengend Snippet: Inhibitions of STIM1 mimics the effects of Phemindole and over expression of STIM1 showed opposite effect. (A) Ca 2+ imaging was performed in control, in the presence of SKF 96365 (SOCE inhibitor) and STIM1 si-RNA treated MDAMB-231 cells. Analog plots of the fluorescence ratio (340/380) of the cells were shown. Knockdown of STIM1 protein was confirmed by western blot analysis (Inset) (B) Quantification (mean ± SD) of fluorescence ratio (340/380). ∗ P < 0.05 versus control. (C) Bar diagram showed MTT assay under SKF 96365, STIM1 si-RNA, treated conditions for 24 h in MDAMB231 cells. (D) STIM1 protein levels were restored by Transfection of STIM1 clones and also other protein levels were measured in presence and absence of Phemindole in STIM1 transfected cells by western blot (left panel). Quantification of band intensity in respect to loading control was shown in bar diagram (left panel). Blots are representative of three independent experiments and quantifications are indicated by bar diagram. Values were normalized by intensity of loading control. (E) Fura-2 Ca 2+ imaging showing the results of a quantitative analysis of Ca 2+ content (or Ca 2+ release in after Tg treatment) and Ca 2+ influx during SOCE as mentioned previously. Values are the mean ± SEM for all cells in plates ∗ p < 0.05 versus control group. (F) Bar diagram represented the cell proliferation pattern of STIM1 over expressed MDAMB-231 cells in presence of Phemindole treatment. Each bar gives the mean ± SEM of three separate experiments. ∗ , ∗∗ , ∗∗∗ , # indicate p < 0.05, p < 0.01, p < 0.0005, p > 0.05 respectively versus untreated or control group.

Article Snippet: MDAMB-231 cells were transfected with 300 pmol of STIM1 siRNA (Santa Cruz Biotechnology) and Lipofectamine 2000 (Invitrogen) separately for 12 h. Levels of STIM1 proteins were estimated by Western blotting.

Techniques: Over Expression, Imaging, Control, Fluorescence, Knockdown, Western Blot, MTT Assay, Transfection, Clone Assay

Journal: Frontiers in Pharmacology

Article Title: Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells

doi: 10.3389/fphar.2016.00114

Figure Lengend Snippet: Assessment of toxicity of Phemindole. (A) 4T1 induced tumors excised from the aforementioned sets were analyzed histopathologically using eosin haematoxylin staining and phase contrast images were captured. (B) Immuno-histochemical analysis of the excised tumors from the untreated and Phemindole treated sets using anti-STIM1 antibody (left panel). Corrected Fluorescence intensity of STIM1 protein was quantified and represented as bar diagram (right panel). (C) Histological sections of liver and kidney from the animals were stained with haematoxylin and counter-stained with eosin and microscopically analyzed for histopathologically examinations of tissue toxicity like cellular damage and vacuolization. (D) Blood was collected after sacrifice the mice from control, untreated tumor and Phemindole treated sets and subsequent Liver function test and Kidney Function Test were performed. All measurements of three groups were repeated at least three times, Values are mean ± SEM of three independent experiments in each case or representative of typical experiment. ∗∗∗ P < 0.001, (E) Schematic diagram depicting the detailed molecular mechanisms of antitumorogenic activity of Phemindole against triple negative breast cancer cells.

Article Snippet: MDAMB-231 cells were transfected with 300 pmol of STIM1 siRNA (Santa Cruz Biotechnology) and Lipofectamine 2000 (Invitrogen) separately for 12 h. Levels of STIM1 proteins were estimated by Western blotting.

Techniques: Staining, Fluorescence, Control, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synaptic crosstalk conferred by a zone of differentially regulated Ca 2+ signaling in the dendritic shaft adjoining a potentiated spine

doi: 10.1073/pnas.1902461116

Figure Lengend Snippet: LTCC activity level governs the spread along the dendritic shaft of STIM1 feedback inhibition of dendritic LTCCs. (A) Mean total cytosolic Ca2+ (RGECO1) changes by uncaging on 79wt neurons at 0 μm. Mean (black) ± SEM (dark gray). Uncaging occurred from 0 to 60 s, at 1 Hz. (B) Mean LTCC-independent Ca2+ changes (IF) in the cytosol by uncaging at 0 μm in the same neurons as A and pretreated with nimodipine (5 μM) for 15 min. Mean (black) ± SEM (red). (C) Mean LTCC-dependent Ca2+ changes (DF) at 0 μm, calculated by subtracting the non-LTCC Ca2+ time course from the total Ca2+ time course of the same neuron. Mean (black) ± SEM (blue). (D) Mean integrated increase in total cytosolic Ca2+ (shaded gray region in A) along a dendrite of 79wt (circles; n = 5), 79ΔPIX (squares; n = 5), and 79ΔPKA (diamonds; n = 6) neurons. Mean ± SEM. (E) Mean integrated increase in the LTCC-independent component of cytosolic Ca2+ (IF; shaded pink region in B) along a dendrite of 79wt (circles; n = 5), 79ΔPIX (squares; n = 5), and 79ΔPKA (diamonds; n = 6) neurons. Mean ± SEM. (F) Mean integrated increase in LTCC-dependent component of cytosolic Ca2+ (DF; shaded light blue region in C) along a dendrite of 79wt (circles; n = 5), 79ΔPIX (squares; n = 5), and 79ΔPKA (diamonds; n = 6) neurons. Mean ± SEM.

Article Snippet: D1ER (Addgene no. 36325), RGECO1 (Addgene no. 32444), and human STIM1-YFP (Addgene no. 19754) were provided by Amy Palmer, University of Colorado, Boulder, CO, Robert Campbell, University of Alberta, Edmonton, Canada, and Anjana Rao, La Jolla Institute for Immunology, La Jolla, CA (via Addgene).

Techniques: Activity Assay, Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synaptic crosstalk conferred by a zone of differentially regulated Ca 2+ signaling in the dendritic shaft adjoining a potentiated spine

doi: 10.1073/pnas.1902461116

Figure Lengend Snippet: β-adrenergic receptors modulate a zone of Ca2+ signaling in the dendritic shaft adjoining a potentiated spine. (A) In neurons treated with Iso, time course of change in total cytosolic Ca2+ (IF + DF, imaged with RGECO1) (n = 5) measured at 0 μm from the uncaging-stimulated spine. Mean (black) ± SEM (dark gray). (B) After 15 min nimodipine treatment, time course of change in LTCC-independent cytosolic Ca2+ (IF) of the neurons in A, measured at 0 μm. Mean (black) ± SEM (red). (C) Time course of increase in LTCC-dependent cytosolic Ca2+ (DF) (DF = total – IF) at 0 μm. Mean (black) ± SEM (blue). (D) Spatial profile for integrated increase in total cytosolic Ca2+ (IF + DF) shaded gray region in A along the dendrite of Iso-treated (1 μM; open circles; n = 5) versus untreated (control) neurons (shaded circles; n = 5) and for Iso-treated + STIM1 knockdown (squares; n = 5). Mean ± SEM. (E) Spatial profile for integrated increase in the LTCC-independent Ca2+ (IF; shaded pink region in B) along the dendrite of Iso-treated (open circles; n = 5) versus untreated (control) neurons (red circles; n = 5) and for Iso-treated + STIM1 knockdown (squares; n = 5). Mean ± SEM. (F) Spatial profile for average integrated increase in LTCC-dependent Ca2+ (DF; shaded light blue region in C) along the dendrite of Iso-treated (open circles; n = 5) versus untreated (control) neurons (blue circles; n = 5) and for Iso-treated + STIM1 knockdown (squares; n = 5). Mean ± SEM.

Article Snippet: D1ER (Addgene no. 36325), RGECO1 (Addgene no. 32444), and human STIM1-YFP (Addgene no. 19754) were provided by Amy Palmer, University of Colorado, Boulder, CO, Robert Campbell, University of Alberta, Edmonton, Canada, and Anjana Rao, La Jolla Institute for Immunology, La Jolla, CA (via Addgene).

Techniques: Control, Knockdown