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: Nature Communications

Article Title: STIM-Orai1 signaling regulates fluidity of cytoplasm during membrane blebbing

doi: 10.1038/s41467-020-20826-5

Figure Lengend Snippet: Membrane blebbing of DLD1 cells expressing either Sec61β-mCherry and GFP-PLCδ-PH ( a , Supplementary Movie ) or Orai1-mCherry and GFP-tagged STIM1 ( b ). Arrowheads show the ER-PM contact sites. Indicated times are relative to the first image. Results shown are representative of three independent experiments. Scale bar, 2 μm. c – f DLD1 cells expressing Lifeact-RFP were treated with the SOCE inhibitors AnCoA4 (50 µM) and SKF96365 (10 µM). c Representative images from three independent experiments. Indicated times are relative to drug treatment. Scale bar, 10 μm. The number ( d , N = 20 cells), area ( e , N = 20 blebs) and retraction velocity ( f , N = 20 blebs) of membrane blebs in vehicle-treated (control) and drug-treated (4-bromo-A23187) DLD1 cells over 10 min from three independent experiments. Individual data points are plotted with the means ± SD. **** P < 0.0001 (One-way ANOVA with Tukey’s post-hoc multiple comparison test). Source data are provided as a Source Data file.

Article Snippet: Following expression vectors were purchased from Addgene; pGP-CMV-GCaMP6s-CAAX (Addgene No.52228), Sec61β-mCherry (Addgene No. 49155), pGP-CMV-GCaMP6s (Addgene No.40753), EGFP-E-Syt1 (Addgene No.66830), GFP-PIP5K gamma (Addgene No. 22299). cDNAs encoding full-length STIM1, Orai1, Mena, VASP, STIMATE, and MRLC1 were amplified by RT-PCR, fused to the sequence encoding EGFP, Scarlet or HA, and ligated into the pCAGGS-neo vector.

Techniques: Expressing

Journal: Nature Communications

Article Title: STIM-Orai1 signaling regulates fluidity of cytoplasm during membrane blebbing

doi: 10.1038/s41467-020-20826-5

Figure Lengend Snippet: a – f Membrane blebbing in DLD1 cells expressing Lifeact-RFP with either WT or dominant negative (E106Q) Orai1. a Representative still images from five independent experiments. b Tricolor maps showing angular coordinates along the horizontal axis and time on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. Results shown are representative of three independent experiments. c Fluorescence intensities of GCaMP6s in “bleb” and “cell body” cytoplasm were quantified in DLD1 cells expressing either WT or E106Q Orai1. The ratio of “bleb” to “cell body” intensities are plotted over time. Data presented are means ± SD based on the values from five independent experiments. The number ( d , N = 20 cells), area ( e , N = 20 blebs), and retraction velocity ( f , N = 10 blebs) of membrane blebs in WT or E106Q Orai1-expressing cells. g – l Membrane blebbing in DLD1 cells expressing Lifeact-RFP with either WT or dominant active (DA, D76A) STIM1. g Representative still images from three independent experiments. Arrowhead indicate persistent ER-PM contact site. h Tricolor maps showing angular coordinates along the horizontal axis and time on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. Results shown are representative of three independent experiments. i Histograms of bleb expansion and retraction velocities in WT an DA STIM1-expressing cells. Three independent measurements are plotted for each condition. The number ( j , N = 20 cells), area ( k , N = 20 blebs), and retraction velocity ( l , N = 10 blebs) of membrane blebs in WT or DA STIM1-expressing cells. Individual data points are plotted with the means ± SD. m Schematic of PM-ER contact site formation in the early stages of bleb formation. Details of the model are described in the text. a and g Scale bar, 10 μm. d – f , j – l **** P < 0.0001 (Two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.

Article Snippet: Following expression vectors were purchased from Addgene; pGP-CMV-GCaMP6s-CAAX (Addgene No.52228), Sec61β-mCherry (Addgene No. 49155), pGP-CMV-GCaMP6s (Addgene No.40753), EGFP-E-Syt1 (Addgene No.66830), GFP-PIP5K gamma (Addgene No. 22299). cDNAs encoding full-length STIM1, Orai1, Mena, VASP, STIMATE, and MRLC1 were amplified by RT-PCR, fused to the sequence encoding EGFP, Scarlet or HA, and ligated into the pCAGGS-neo vector.

Techniques: Expressing, Dominant Negative Mutation, Fluorescence

Journal: Nature Communications

Article Title: STIM-Orai1 signaling regulates fluidity of cytoplasm during membrane blebbing

doi: 10.1038/s41467-020-20826-5

Figure Lengend Snippet: a Representative still images of membrane blebbing in DLD1 cells expressing Lifeact-RFP and GFP-E-Syt1 from five independent experiments. See also Supplementary Movie . b , c DLD1 cells expressing Lifeact-RFP with either GFP-E-Syt1 ( b ) or GCaMP6s ( c ) were treated with the actin polymerization inhibitor Latrunculin B (LatB, 10 µM). Arrowheads show disrupted actin cortex. Results shown are representative of three independent experiments. See also Supplementary Movie . d – f Biochemical analyses of ezrin’s role in STIM1-Orai1 complex formation. d Orai1-HA was co-expressed with either GFP or GFP-ezrin. Inputs and HA immunoprecipitates were immunoblotted with antibodies against HA and GFP. e STIM1-FLAG and Orai1-HA were co-expressed with either GFP or GFP-ezrin. Inputs and FLAG immunoprecipitates were immunoblotted with antibodies against FLAG, HA, and GFP. f GFP expression levels (shaded in green) and co-precipitated Orai1-HA (individual measurements) were quantified and normalized to GFP-expressing control. Data presented are means ± SD based on the values from four independent experiments. g , h Proximity ligation assay (PLA) for in situ detection of Orai1-STIM1 interaction. Representative images from five independent experiments are shown in g . h Quantification of PLA signals in expanding (low Lifeact intensity) and retracting (high Lifeact intensity) blebs based on data shown in g and Supplementary Fig. . STIMATE-STIM1 and Claudin-3-STIM1 are positive and negative controls, respectively. N = 25 independent blebs. Individual data points are plotted with the means ± SD. i Membrane blebbing in Ezrin KO cells expressing GFP- PLCδ-PH and Sec61β-mCherry (upper panels) and in WT DLD1 cells expressing constitutive active Rnd3 (S240A) and Sec61β-mCherry (lower panels). Arrowheads indicate persistent ER-PM contact sites. Results shown are representative of three independent experiments. j Representative images from three independent experiments of DLD1 cells expressing constitutive active Ezrin (T567E) and Sec61β-mCherry. a , g (middle and bottom panels), i , j (three right panels) Scale bar, 2 μm. b , c , g (top panels), and j (left panel) Scale bar, 10 μm. f , h **** P < 0.0001 (Two-sided, unpaired Student’s t test). Times shown are relative to drug treatment ( b, c ) or the first image ( i ). Source data are provided as a Source Data file.

Article Snippet: Following expression vectors were purchased from Addgene; pGP-CMV-GCaMP6s-CAAX (Addgene No.52228), Sec61β-mCherry (Addgene No. 49155), pGP-CMV-GCaMP6s (Addgene No.40753), EGFP-E-Syt1 (Addgene No.66830), GFP-PIP5K gamma (Addgene No. 22299). cDNAs encoding full-length STIM1, Orai1, Mena, VASP, STIMATE, and MRLC1 were amplified by RT-PCR, fused to the sequence encoding EGFP, Scarlet or HA, and ligated into the pCAGGS-neo vector.

Techniques: Expressing, Proximity Ligation Assay, In Situ

Journal: Journal of Cell Science

Article Title: Imaging elemental events of store-operated Ca 2+ entry in invading cancer cells with plasmalemmal targeted sensors

doi: 10.1242/jcs.224923

Figure Lengend Snippet: Targeting the Ca2+ biosensor GCaMP6f to the plasma membrane. (A) Schematic showing the design strategy of the GCaMP6f-Orai1 Ca2+ biosensor. GCaMP6f was fused to the N-terminus of Orai1 (GCaMP6f-Orai1) to target it to the plasma membrane with preferential accessibility to Orai1-mediated Ca2+ entry. This vector was expressed in WM793 cells by adenoviral infection. (B) Localization of GCaMP6f-Orai1. Upper panel: confocal images of GCaMP6f-Orai1 (top left), invadopodia visualized by Lifeact-mApple fluorescence (top middle) or degradation holes (arrowheads) left after TRITC-labeled gelatin was digested (bottom middle, an enlarged view of the cell area is indicated in the lower panel), and their merged images. Note that the GCaMP6f-Orai1 fluorescence encircles invadopodia. Lower panel: orthogonal views of confocal z stacks, showing the localization of the targeted sensor: XY view (left), YZ view (middle) and an enlarged view of the boxed area indicated in the YZ view (right). (C) Western blots (left) showing the expression of GCaMP6f-Orai1 and its effect on endogenous STIM1 and Orai1 levels, and quantification (right). (D) Effects of biosensor expression on thapsigargin (TG)-induced SOCE. Left: after store Ca2+ release induced by 5 μM TG in 0 Ca2+ solution, reintroducing Ca2+ into the extracellular solution elicited SOCE. Note that Rhod4 indicated similar release and SOCE-induced cytosolic Ca2+ responses in control and GCaMP6f-Orai1-expressing cells. Right: statistics of the magnitude of SOCE, indexed by ΔF/F0 of Rhod4 after Ca2+ restoration following store depletion. n=172 and 49 cells from 7 independent experiments for control and GCaMP6f-Orai1 groups, respectively. (E) The number of invadopodia per cell was unaltered by biosensor expression. n=62 cells from 4 independent experiments for both groups. Data represent mean±s.e.m.; NS, not statistically significant.

Article Snippet: Antibodies against STIM1 (Cell Signaling Technology, #5668S), Orai1 (Sigma-Aldrich, #O8264), GAPDH (Sigma-Aldrich, #G8795), Src (Millipore, clone GD11, #05-184), p-Src (Tyr416) (Cell Signaling Technology, #2101), Pyk2 (Cell Signaling Technology, #3480), p-Pyk2 (Tyr402) (Cell Signaling Technology, #3291), tubulin (Sigma-Aldrich, #T6199), calmodulin (Santa Cruz Biotechnology, #sc-137079) and TRPM7 (Abcam, #ab109438) were used.

Techniques: Clinical Proteomics, Membrane, Plasmid Preparation, Infection, Fluorescence, Labeling, Western Blot, Expressing, Control

Journal: Journal of Cell Science

Article Title: Imaging elemental events of store-operated Ca 2+ entry in invading cancer cells with plasmalemmal targeted sensors

doi: 10.1242/jcs.224923

Figure Lengend Snippet: Invadopodial Ca2+ glows originate from SOCE mediated by Orai1 and STIM1. (A,B) Manipulating SOCE in WM793 cells. Western blots showing the expression of the dominant-negative mutant Orai1-E106A (E106A) (A) and knockout of STIM1 (STIM1 KO) (B). (C) Representative traces (left) and average amplitude (right) of SOCE recorded by Fluo4. 100 μM 2-APB was added to the extracellular solution when the fluorescence returned to baseline after TG treatment. Note that ER Ca2+ release was largely comparable in these groups (n=69, 32, 61 and 83 cells from 3, 3, 9 and 3 independent experiments for control, 2-APB, E106A and STIM1 KO groups, respectively). (D) Statistics of the frequencies of spontaneous invadopodial Ca2+ glows (n=54, 18, 21 and 36 cells from 30, 8, 16 and 25 independent experiments for control, 2-APB, E106A mutant and STIM1 KO groups, respectively). (E) Depression of invadopodial formation by treatment with 2-APB (n=66), expression of the E106A mutant (n=52) and STIM1 KO (n=57) compared with control (n=62) from 6 independent experiments. (F) Invadopodial Ca2+ glows resolved during global SOCE. Top: space-time plots of local Ca2+ events reported by GCaMP6f-Orai1 in response to 0 Ca, TG and subsequent 2 mM Ca2+ in a WM793 cell. The colored peaks are overlays of surface plots of discrete Ca2+ glows at their peaks, indicating both the locations and the amplitudes (coded by colors) of these local signals. Bottom: corresponding traces of representative invadopodial Ca2+ glows during global SOCE. Note the initial near-synchronous activation at all invadopodia followed by delayed, asynchronous activity at individual invadopodia. (G–I) Invadopodial Ca2+ glows were abolished by 2-APB treatment (G), expression of the E106A mutant (H) and STIM1 KO (I). Data are presented as mean±s.e.m.; ***P<0.001 compared with control.

Article Snippet: Antibodies against STIM1 (Cell Signaling Technology, #5668S), Orai1 (Sigma-Aldrich, #O8264), GAPDH (Sigma-Aldrich, #G8795), Src (Millipore, clone GD11, #05-184), p-Src (Tyr416) (Cell Signaling Technology, #2101), Pyk2 (Cell Signaling Technology, #3480), p-Pyk2 (Tyr402) (Cell Signaling Technology, #3291), tubulin (Sigma-Aldrich, #T6199), calmodulin (Santa Cruz Biotechnology, #sc-137079) and TRPM7 (Abcam, #ab109438) were used.

Techniques: Western Blot, Expressing, Dominant Negative Mutation, Knock-Out, Fluorescence, Control, Mutagenesis, Activation Assay, Activity Assay

Journal: Journal of Cell Science

Article Title: Imaging elemental events of store-operated Ca 2+ entry in invading cancer cells with plasmalemmal targeted sensors

doi: 10.1242/jcs.224923

Figure Lengend Snippet: SOCE regulates invadopodial formation through the Ca2+/calmodulin–Pyk2–Src pathway. (A) STIM1 shRNA and Orai1 shRNA decreased active Pyk2 (pY402-Pyk2) levels in WM793 cells. (B) Ectopic expression of STIM1- and Orai1-activated Pyk2 in WM793 cells. (C) SOCE blockade with 2-APB (100 µM) decreased pY402-Pyk2 levels. (D) Depletion of Pyk2 with shRNA abrogated the STIM1-mediated activation of pY416-Src. (E) Immunofluorescence staining of pY402-Pyk2, showing that Pyk2 was activated in invadopodia and focal adhesions in WM793 cells. (F) Immunofluorescence staining showing that calmodulin was recruited to invadopodia in WM793 cells. (G) Pyk2 knockdown abolished STIM1-mediated invadopodial formation (n=120, 123, 125 and 127 for control shRNA, control shRNA+STIM1, Pyk2 shRNA, and Pyk2 shRNA+STIM1, respectively). (H) STIM1-promoted WM793 cell invasion was abrogated after Pyk2 knockdown. Cell numbers were collected from triplicate independent invasion chamber assays. (I) Ectopic overexpression of STIM1 stimulated the interaction of Src with wild-type Pyk2, but not the calmodulin-binding motif mutant of Pyk2 (L176Q/Q177A). (J) A model for the Ca2+/calmodulin–Pyk2–Src pathway downstream of SOCE. Local high Ca2+ influx mediated by SOCE promotes the binding of Ca2+/calmodulin to the FERM domain of Pyk2, which releases the intramolecular auto-inhibition. Ca2+/calmodulin-bound Pyk2 autophosphorylate each other on tyrosine 402. pY402-Pyk2 interacts with the SH2 domain of Src to activate Src. Data are representative results from at least 3 independent experiments. Horizontal bars represent mean±s.e.m; ***P<0.001, **P<0.01; NS, not statistically significant.

Article Snippet: Antibodies against STIM1 (Cell Signaling Technology, #5668S), Orai1 (Sigma-Aldrich, #O8264), GAPDH (Sigma-Aldrich, #G8795), Src (Millipore, clone GD11, #05-184), p-Src (Tyr416) (Cell Signaling Technology, #2101), Pyk2 (Cell Signaling Technology, #3480), p-Pyk2 (Tyr402) (Cell Signaling Technology, #3291), tubulin (Sigma-Aldrich, #T6199), calmodulin (Santa Cruz Biotechnology, #sc-137079) and TRPM7 (Abcam, #ab109438) were used.

Techniques: shRNA, Expressing, Activation Assay, Immunofluorescence, Staining, Knockdown, Control, Over Expression, Binding Assay, Mutagenesis, Inhibition