Journal: Cell reports

Article Title: Altered GM1 catabolism affects NMDAR-mediated Ca 2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model

doi: 10.1016/j.celrep.2024.114117

Figure Lengend Snippet: (A) Immunoblot analysis of cell fractions from 6-month-old WT and β-Gal − / − mice. Markers for the ER (Calnexin), PM (N-cadherin), and ER-PM junctions (ORAI1, STIM1, STIM2, VAPA, and VAPB) were enriched in their respective fractions. Immunoblots using HRP-conjugated cholera toxin B subunit (CTX-B) show high GM1 levels in β-Gal − / − fractions. (B) Representative HPTLC plate showing GM1 levels in the ER, PM, and ER-PM junctions isolated from 6-month-old WT and β-Gal − / − mice. STD, standard. Note: to detect GM1 in WT samples, the sample volume loaded was 3× that of the β-Gal − / − samples. (C) Quantification of GM1 levels from HPTLC plates performed in (B). n = 8. Values are expressed as median ± quartiles. Statistical analysis was performed using the Student’s t test; *** p < 0.001, **** p < 0.0001. (D) Representative HPTLC plate showing GM1 levels in ER-PM junctions isolated from 1-, 3-, and 6-month-old WT and β-Gal − / − mice. To detect GM1 in WT samples, the sample volume loaded was 3× that of the β-Gal − / − samples. (E) Quantification of GM1 levels from HPTLC plates performed in (D). n = 4. Values are expressed as median ± quartiles. Statistical analysis was performed using the Student’s t test with Welch’s correction; ns, not significant; *** p < 0.001, **** p < 0.0001.

Article Snippet: Rabbit anti-STIM1 antibody , Cell Signaling , Cat #: 4916; RRID: AB_2271287.

Techniques: Western Blot, High Performance Thin Layer Chromatography, Isolation

Journal: Cell reports

Article Title: Altered GM1 catabolism affects NMDAR-mediated Ca 2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model

doi: 10.1016/j.celrep.2024.114117

Figure Lengend Snippet:

Article Snippet: Rabbit anti-STIM1 antibody , Cell Signaling , Cat #: 4916; RRID: AB_2271287.

Techniques: Virus, Recombinant, Isolation, Magnetic Beads, Plasmid Preparation, Software

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: PLoS ONE

Article Title: NK Cells Respond to Haptens by the Activation of Calcium Permeable Plasma Membrane Channels

doi: 10.1371/journal.pone.0151031

Figure Lengend Snippet: (A ) Ca 2+ flux induced by Bourgeonal, Oxa and DNFB in the presence of 2-APB (100 μM) and BTP2 (10 μM) in NK cells. The response by the odorant/hapten is shown in red, that in the presence of 2-APB is in blue, and that of BTP2 in yellow. The bar graph depicts the mean fold change (±SEM) in Indo-1 emission (i.e. the Indo-1V/Indo-1B ratio observed at the peak response relative to that of the baseline) induced by the stimulus alone (black bar) or the stimulus plus 2-APB (dark grey bar) or BTP2 (light grey) in NK cells from naive mice. n = 3 independent experiments, statistics as compared to stimulus alone using t-test ** p<0.01, * p<0.05, ns not significant (p>0.05). ( B ) Ca 2+ flux induced by Bourgeonal, Oxa and DNFB in the presence of 2-APB (100 μM) and BTP2 (10 μM) in Jurkat cells ( B ). Representative of 2–3 independent experiments. (C ) HEK293 cells were stably transfected with hSTIM1 without or with hORAI1, hORAI2 or hORAI3. Transfectants were kept in Ca 2+ -free medium and intracellular Ca 2+ stores were depleted using Thapsigargin (TG) (1 μM). Ca 2+ entry was measured following the addition of extracellular CaCl 2 (1 mM). Maximal Ca 2+ entry was detected when HEK293 cells co-expressed hSTIM1 plus hORAI1, hORAI2 or hORAI3. (F ) Oxa (0.4 mM) and DNFB (0.25 mM) fail to induce Ca 2+ flux in hSTIM1/hORAI transfected HEK293 cells.

Article Snippet: Yasuo Mori, Kyoto University), pcDNA3.1-ORAI1 (Addgene #21638), ORAI2 (#16369), ORAI3 (#16370) and human STIM1-YFP (#19754) using calcium-phosphate transfection.

Techniques: Stable Transfection, Transfection

Journal: Neuron

Article Title: Axonal endoplasmic reticulum Ca 2+ content controls release probability in CNS nerve terminals

doi: 10.1016/j.neuron.2017.01.010

Figure Lengend Snippet: (A-B) Neurons expressing ER-GCaMP6-150 and cytosolic jRCaMP1b were co-transfected with or without an shRNA targeting STIM1 and were used to examine single AP-driven Ca2+ influx (jRCaMP1b) and axonal [Ca2+]ER (ER-GCaMP6-150) in the same cells. Recordings from wild type (n=15) and STIM1 KD (n=12) neurons were grouped using a binning size of [Ca2+]ER=25µM to better estimate fitting parameters (see methods; see Fig. S6 for unbinned data). These two variables are correlated only in wild type cells (A) and are well described by a generalized Hill equation (red line) with a K1/2 of 105 µM ± 5 µM and a Hill coefficient of 5.4 ± 2.7(Adjusted R-square = 0.87). Fitting in the case of STIM1 KD was not possible (B, see methods). Grey and brown lines show the predicted impact of CPA on Ca2+ influx when the average value of [Ca2+]ER (152 µM) decreases by 48 µM, as measured during SERCA block.

Article Snippet: To quantify endogenous STIM1 expression and measure knockdown efficiency, primary neurons expressing cytosolic GFP alone or in combination with STIM1 shRNA vector were analyzed by immunocytochemistry: after 4% PFA fixation, neurons were blocked and permeabilized for 90 min at room temperature in 0.3% Triton X-100, 10% normal goat serum and 1% BSA, and stained with antibodies overnight at 4°C against STIM1 (rabbit, Cell Signaling, #5668; 1:800) and GFP (chicken, Life Technologies, # {"type":"entrez-nucleotide","attrs":{"text":"A10262","term_id":"490678"}} A10262 , 1:1000).

Techniques: Expressing, Transfection, shRNA, Blocking Assay

Journal: Neuron

Article Title: Axonal endoplasmic reticulum Ca 2+ content controls release probability in CNS nerve terminals

doi: 10.1016/j.neuron.2017.01.010

Figure Lengend Snippet: (A-D) Single AP-driven cytosolic Ca2+ signals and exocytosis were measured using GCaMP6f or vG-pH, respectively. Single-AP stimulated signals were quantified in wild type neurons, STIM1 KD neurons, STIM1 KD neurons expressing an shRNA-resistant version of STIM1 (STIM1WT) or STIM1 KD neurons expressing an shRNA-resistant version of STIM1 that is insensitive to ER Ca2+ content due to EF-hand mutations (STIM1EF). Responses were quantified before (black) and after (red) CPA treatment. Black dashed lines represent average response before treatment whereas red dashed lines represent the effect quantified in wild type neurons for ease of comparison in the different conditions. (C-D) Differential effects of CPA are summarized by showing the remaining response after CPA treatment in each of the conditions. Solid black line indicates response before CPA treatment, normalized to 1 in each case. (C) Effects of CPA in single-AP-driven presynaptic Ca2+ signals, Control n=16, ***p=0.0016; STIM1 KD n=14, n.s. p=0.60; STIM1 KD + STIM1WT n=10, *p=0.02; STIM1 KD + STIM1EF n=10, n.s. p=0.14. (D) Effects of CPA in single-AP vG-pH peak responses, Control n=10, ***p=6.68·10−5; STIM1 KD n=7, n.s. p=0.22; STIM1 KD + STIM1WT n=7, *p=0.031; STIM1 KD + STIM1EF n=7, n.s. p=0.55. Statistics were analyzed using paired sample Student’s t-test.

Article Snippet: To quantify endogenous STIM1 expression and measure knockdown efficiency, primary neurons expressing cytosolic GFP alone or in combination with STIM1 shRNA vector were analyzed by immunocytochemistry: after 4% PFA fixation, neurons were blocked and permeabilized for 90 min at room temperature in 0.3% Triton X-100, 10% normal goat serum and 1% BSA, and stained with antibodies overnight at 4°C against STIM1 (rabbit, Cell Signaling, #5668; 1:800) and GFP (chicken, Life Technologies, # {"type":"entrez-nucleotide","attrs":{"text":"A10262","term_id":"490678"}} A10262 , 1:1000).

Techniques: Expressing, shRNA

Journal: Neuron

Article Title: Axonal endoplasmic reticulum Ca 2+ content controls release probability in CNS nerve terminals

doi: 10.1016/j.neuron.2017.01.010

Figure Lengend Snippet: (A) Representative image of an individual axon expressing STIM1-RFP and the presynaptic marker synapsin-GFP (straightened for ease of visualization). (B-E) Synapsin-GFP and STIM1-RFP were imaged before (B) and after CPA treatment (C). (D-F) Intensity profiles of both proteins were analyzed in 218 individual boutons from 8 neurons and compared before and after 15 min of CPA treatment to obtain 1 dimensional intensity cross-correlation profiles, showing that the presynaptic enrichment of STIM1-RFP increases following CPA treatment (F; control=0.66 ± 0.03, CPA=0.75 ± 0.02; ** p=0.0012) with negligible impact on the distribution of synapsin-GFP (control, black profile in D; CPA, blue profile in E). (G) Average decrease in single AP-Ca2+ influx following overexpression of STIM1-RFP. (H) Representative image of a neuron co-transfected with Phy-GCaMP6 (top, left) and STIM1-RFP (bottom left) showing the response to a 10 AP stimulus (middle left). Note that nerve terminals with greater STIM1-RFP abundance (1) have lower Ca2+ influx than neighboring synapses (2,3) with lower STIM1-RFP abundance (right) (I) Quantification of 10AP-driven phy-GCaMP6 signals shown in H. (J) Quantification of single AP-driven phy-GCaMP6 signals and their corresponding STIM1-RFP intensities acquired from 564 individual boutons from 10 neurons co-transfected as in H. STIM1-RFP intensities are shown as fluorescence normalized to the auto-fluorescence. Phy-GCaMP6 responses were binned into 4 different STIM1-RFP intensity groups (1–1.5, 1.5–2.5, 2.5–4.5 and >4.5) and reveal a strong inverse correlation between STIM1-RFP abundance and Ca2+ influx.

Article Snippet: To quantify endogenous STIM1 expression and measure knockdown efficiency, primary neurons expressing cytosolic GFP alone or in combination with STIM1 shRNA vector were analyzed by immunocytochemistry: after 4% PFA fixation, neurons were blocked and permeabilized for 90 min at room temperature in 0.3% Triton X-100, 10% normal goat serum and 1% BSA, and stained with antibodies overnight at 4°C against STIM1 (rabbit, Cell Signaling, #5668; 1:800) and GFP (chicken, Life Technologies, # {"type":"entrez-nucleotide","attrs":{"text":"A10262","term_id":"490678"}} A10262 , 1:1000).

Techniques: Expressing, Marker, Over Expression, Transfection, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca 2+ /NFATc3 Signaling

doi: 10.3389/fcell.2021.779373

Figure Lengend Snippet: Ttyh1 is expressed in Ki67 - quiescent NSCs in SVZ. (A) Double-labeling of Ttyh1 and NSC markers. Ttyh1 co-labeled cells with CD133, GFAP and Sox2 at high frequency in SVZ. (B) Double-labeling of Ttyh1 and TAP or neuroblast cell markers. Ttyh1 seldomly labeled EGFR + and DCX + cells. (C) Double-labeling of Ttyh1 and proliferative cell marker Ki67. Ttyh1 completely were not co-labeled with Ki67 in entire SVZ region. The non-overlapping of Ttyh1 + and Ki67 + signals are showed in magnifications within white rectangles (the lower field is the magnification of the dotted rectangle in upper field). (D) Statistics of the proportions of co-labeled cells in Ttyh1 + cells. Scale bar = 10 μm in A, Scale bar = 50 μm in B, Scale bar = 100 μm in C, Scale bar = 10 μm in D. n = 3 for all experiments. Data are expressed as mean ± SEM. LV, lateral ventricle.

Article Snippet: Membranes were blocked using 5% skim milk at room temperature for 1 h, and incubated at 4°C overnight with the addition of the primary antibodies of β-actin (1:10,000, Abcam), Ttyh1 (1:400, self-prepared , STIM1 (1:400, Proteintech, Chicago, IL, United States), Orai1 (1:400, Proteintech), NFATc3 (1:500, Proteintech), CALM (1:1,000, Abcam), CaMKII (1:1,000, Abcam), Cyclin D1 (1:5,000, Proteintech), p21 (1:1,000, Abcam).

Techniques: Labeling, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca 2+ /NFATc3 Signaling

doi: 10.3389/fcell.2021.779373

Figure Lengend Snippet: Ttyh1 is mainly distributed in qNSCs and astrocytes in neurogenic niche. (A) Diagrams of Ttyh1-CreERT2 mice construction and tamoxifen induction strategy. Targeting vector introduced P2A-iCreERT2 into the 13th exon located at the 3’ terminal of Ttyh1 gene to construct Ttyh1-CreERT2 mice, with Ttyh1 ORF intact. After crossing with Ai9 Rosa26-tdTomato reporter mice, Ttyh1-CreERT2; Rosa-tdTomato mice were obtained and were injected tamoxifen for continuous 5 days at 8 weeks old. Samples were taken at 5 days post induction (dpi) and 10 dpi for analysis. (B) Immunofluorescence analysis of reporter mice in neurogenic niches. The samples taken from 5 dpi group showed that Ttyh1-tomato + cells in SVZ were mainly concentrated on the striatal side (arrowheads) and dorsal wedge (arrows). These Ttyh1-tomato + cells mainly co-labeled with Sox2 and GFAP, and less co-labeled with DCX in SVZ and SGZ. Ttyh1-tomato + cells also co-labeled with EGFR and γ-tubulin at a small proportion in SVZ. (C) Statistics of the proportions of co-labeled cells in Ttyh1-tomato + cells. The proportions of Sox2 + Ttyh1 + cells and GFAP + Ttyh1 + cells were significantly reduced at 10 dpi compared with those at 5 dpi. (D) Single-cell cluster analysis of single-cell RNA sequencing data showed the classification of adult NSCs in SVZ and their progeny cells. (E) The parallel analysis of D showed the distribution of Ttyh1 transcripts in cell clusters. Purple dots represent Ttyh1 + cells. (F) Violin plot of Ttyh1 distribution in NSCs and progeny cells based on single-cell RNA sequencing data. Ttyh1 was highly expressed in qNSCs and astrocytes (in accordance with accumulated Tomato + cells in SVZ, and scattered Tomato + cells outside SVZ in B, respectively). Scale bar = 50 μm in B. n = 3 for all experiments. Data are expressed as mean ± SEM. Statistical significance was calculated using an unpaired, two-tailed Student’s t -test. (∗∗) p < 0.01, (∗) p < 0.05. LV, lateral ventricle.

Article Snippet: Membranes were blocked using 5% skim milk at room temperature for 1 h, and incubated at 4°C overnight with the addition of the primary antibodies of β-actin (1:10,000, Abcam), Ttyh1 (1:400, self-prepared , STIM1 (1:400, Proteintech, Chicago, IL, United States), Orai1 (1:400, Proteintech), NFATc3 (1:500, Proteintech), CALM (1:1,000, Abcam), CaMKII (1:1,000, Abcam), Cyclin D1 (1:5,000, Proteintech), p21 (1:1,000, Abcam).

Techniques: Plasmid Preparation, Construct, Injection, Immunofluorescence, Labeling, RNA Sequencing, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca 2+ /NFATc3 Signaling

doi: 10.3389/fcell.2021.779373

Figure Lengend Snippet: Ttyh1 maintains the stemness of NSCs in vitro . (A) Bright field microscopy showed the morphological disparity between NSCs transfected with negative control shRNA (NC) and shTtyh1. We added NC and shTtyh1 containing lentiviruses respectively on primary cultured NSCs. After 48 h of transfection, the cells in the control group aggregated to form neurospheres and grew in suspension, while the cells in the shTtyh1 group protruded neurites and adhered to the plate. (B) Immunofluorescence staining of NSCs after transfection. After 72 h of transfection, the NSCs were collected and resuspended into single cell suspensions, plated on cover glass and incubated for 24 h, and then subjected to immunofluorescence staining. Compared with the control group, the proportions of Sox2 + cells (left) and EGFR + cells (middle) in the shTtyh1 group were significantly reduced, whereas DCX + cells (right) were not significantly changed. (C) Statistics of the percentages of immunopositive cells in B. (D) Immunofluorescence staining of differentiated neural cell markers. Compared with the control group, the proportions of GFAP + and O4 + cells were increased, whereas the proportion of Map2 + cells were decreased. (E) Statistics of the percentages of immunopositive cells in D. Scale bar = 50 μm in A, Scale bar = 200 μm in B, D. n = 5 for all experiments. Data are expressed as mean ± SEM. Statistical significance was calculated using an unpaired, two-tailed Student’s t -test. (∗∗∗) p < 0.001, (∗∗) p < 0.01.

Article Snippet: Membranes were blocked using 5% skim milk at room temperature for 1 h, and incubated at 4°C overnight with the addition of the primary antibodies of β-actin (1:10,000, Abcam), Ttyh1 (1:400, self-prepared , STIM1 (1:400, Proteintech, Chicago, IL, United States), Orai1 (1:400, Proteintech), NFATc3 (1:500, Proteintech), CALM (1:1,000, Abcam), CaMKII (1:1,000, Abcam), Cyclin D1 (1:5,000, Proteintech), p21 (1:1,000, Abcam).

Techniques: In Vitro, Microscopy, Transfection, Negative Control, shRNA, Cell Culture, Control, Suspension, Immunofluorescence, Staining, Incubation, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca 2+ /NFATc3 Signaling

doi: 10.3389/fcell.2021.779373

Figure Lengend Snippet: Loss of Ttyh1 leads to enhanced neurogenesis in adult mice. (A,B) Immunolabeling of cell-type-specific markers and quantification of immunopositive cells in the adult SVZ. The BrdU retaining cells, Sox2 + GFAP + NSCs, EGFR + TAPs, DCX + neuroblasts and CD24 ependymal cells were compared (A) and quantified (B) in SVZ between control and Ttyh1 KO mice at 2-months old (2 M). (C,D) Immunolabeling of cell-type-specific markers and quantification of immunopositive cells in the adult SGZ. The BrdU retaining cells, Sox2 + GFAP + NSCs, and DCX + neuroblasts were compared (C) and quantified (D) in SGZ between control and Ttyh1 KO mice at 2 M. (E) Open field and Morris water maze tests between control and Ttyh1 knockout mice. Scale bar = 100 μm in A, C. n ≥ 4 for all experiments. Data are expressed as mean ± SEM. Statistical significance was calculated using an unpaired, two-tailed Student’s t -test. (∗∗∗) p < 0.001, (∗∗) p < 0.01, (∗) p < 0.05.

Article Snippet: Membranes were blocked using 5% skim milk at room temperature for 1 h, and incubated at 4°C overnight with the addition of the primary antibodies of β-actin (1:10,000, Abcam), Ttyh1 (1:400, self-prepared , STIM1 (1:400, Proteintech, Chicago, IL, United States), Orai1 (1:400, Proteintech), NFATc3 (1:500, Proteintech), CALM (1:1,000, Abcam), CaMKII (1:1,000, Abcam), Cyclin D1 (1:5,000, Proteintech), p21 (1:1,000, Abcam).

Techniques: Immunolabeling, Control, Knock-Out, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca 2+ /NFATc3 Signaling

doi: 10.3389/fcell.2021.779373

Figure Lengend Snippet: Ttyh1 prevents accelerated exhaustion of the elderly NSCs pool. (A) Immunofluorescence staining of brain sections of elder control and Ttyh1 KO mice at 12-months old (12 M). Sox2 + GFAP + NSCs, EGFR + TAPs, DCX + neuroblasts, Ki67 proliferating cells and CD24 ependymal cells were compared in neurogenic niche between control and Ttyh1 KO mice. (B) Statistics of immunofluorescence staining results in A. Scale bar = 50 μm in A. n = 4 for all experiments. Data are expressed as mean ± SEM. Statistical significance was calculated using an unpaired, two-tailed Student’s t -test. (∗) p < 0.05.

Article Snippet: Membranes were blocked using 5% skim milk at room temperature for 1 h, and incubated at 4°C overnight with the addition of the primary antibodies of β-actin (1:10,000, Abcam), Ttyh1 (1:400, self-prepared , STIM1 (1:400, Proteintech, Chicago, IL, United States), Orai1 (1:400, Proteintech), NFATc3 (1:500, Proteintech), CALM (1:1,000, Abcam), CaMKII (1:1,000, Abcam), Cyclin D1 (1:5,000, Proteintech), p21 (1:1,000, Abcam).

Techniques: Immunofluorescence, Staining, Control, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca 2+ /NFATc3 Signaling

doi: 10.3389/fcell.2021.779373

Figure Lengend Snippet: Transcriptome sequencing results after Ttyh1 knockdown indicate involvement of Calcium signaling and cell cycle genes. (A) Differential gene volcano plot. Red dots represent 998 up-regulated genes, and blue dots represent 409 down-regulated genes. We used false discovery rate (FDR) < 0.05 and fold change ≥2 as criteria to screen for genes with significant differences. (B) Heatmap of the top 30 genes with the most significant difference in up-regulation and down-regulation, respectively. Among them, cell cycle gene Ccnd1 (Cyclin D1) was significantly up-regulated in the Ttyh1 knockdown group. (C) Gene Ontology (GO) analysis. Colors represent q values on a log scale (with red corresponding to the most highly significant). Node size represents the number of genes in a category. Top 20 items of q value are listed. (D) KEGG pathway analysis. The vertical coordinate is −log10 (q value), and the horizontal coordinate is Z-score value (the proportion of the difference between the number of up-regulated genes and the number of down-regulated genes in the total differential genes), and the yellow line represents the threshold of p = 0.05. On the right is a list of signal pathways with the top 20 p -values, with Calcium signaling mostly significant. Different colors represent different categories. Green represents environmental information processing, purple represents cellular processes, red represents human diseases, yellow represents organismal systems, and blue represents metabolism. (E) Heatmap of stage-specific markers of NSCs between control and Ttyh1 knockdown groups. (F) GSEA (gene set enrichment analysis). After Ttyh1 knockdown, calcium signaling pathway and cell cycle related genes were interfered.

Article Snippet: Membranes were blocked using 5% skim milk at room temperature for 1 h, and incubated at 4°C overnight with the addition of the primary antibodies of β-actin (1:10,000, Abcam), Ttyh1 (1:400, self-prepared , STIM1 (1:400, Proteintech, Chicago, IL, United States), Orai1 (1:400, Proteintech), NFATc3 (1:500, Proteintech), CALM (1:1,000, Abcam), CaMKII (1:1,000, Abcam), Cyclin D1 (1:5,000, Proteintech), p21 (1:1,000, Abcam).

Techniques: Sequencing, Knockdown, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca 2+ /NFATc3 Signaling

doi: 10.3389/fcell.2021.779373

Figure Lengend Snippet: Ttyh1 is involved in the regulation of calcium signaling in NSCs. (A) RT-qPCR analysis showed the expression levels of SOCE regulatory molecules between control and Ttyh1 KO neurospheres at primary culture (P0) and Passage 2 (P2). The RNA expressions of STIM1, STIM2, and Orai1 increased at P0, and decreased at P2 generation. (B) Western blotting analysis showed the protein levels of STIM1 and Orai1 between control and Ttyh1 KO neurospheres in P2 generation. (C,D) Intracellular calcium measurement between control and Ttyh1 KO NSCs. We used Flu8 to label the calcium ions in NSCs, then add 10 μM Thapsigargin (TG) to induce store-Ca 2+ release from the endoplasmic reticulum, and finally add 2 μM calcium chloride solution to detect the change of store-operated Ca 2+ entry (SOCE). The results showed that both store-Ca 2+ release and SOCE reduced significantly in P2 generation of Ttyh1 knockout NSCs. (E) Western blotting analysis of downstream molecules of Calcium signaling pathway. Representative Western blots (left) and quantification of bands intensities (right) are present. After Ttyh1 was knockdown by shRNA containing lentivirus, the protein levels of CALM, CaMKII, NFATc3, and p21 were decreased, and that of Cyclin D1 increased significantly. n ≥ 4 for all experiments. Data are expressed as mean ± SEM. Statistical significance was calculated using an unpaired, two-tailed Student’s t -test. (∗) (∗∗∗∗) p < 0.0001, (∗∗∗) p < 0.001, (∗∗) p < 0.01, (∗) p < 0.05.

Article Snippet: Membranes were blocked using 5% skim milk at room temperature for 1 h, and incubated at 4°C overnight with the addition of the primary antibodies of β-actin (1:10,000, Abcam), Ttyh1 (1:400, self-prepared , STIM1 (1:400, Proteintech, Chicago, IL, United States), Orai1 (1:400, Proteintech), NFATc3 (1:500, Proteintech), CALM (1:1,000, Abcam), CaMKII (1:1,000, Abcam), Cyclin D1 (1:5,000, Proteintech), p21 (1:1,000, Abcam).

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot, Knock-Out, Knockdown, shRNA, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca 2+ /NFATc3 Signaling

doi: 10.3389/fcell.2021.779373

Figure Lengend Snippet: NFATc3 is a key transcription factor in the regulation of cell cycle by Ttyh1. (A) RT-qPCR analysis showed the expression levels of five NFAT family members between control and Ttyh1 KO neurospheres at primary culture (P0) and Passage 2 (P2). The RNA expression of NFATc3 increased at P0 and decreased significantly at P2. (B) Western blotting analysis showed the protein level of NFATc3 was also increased at P0 but decreased at P2. (C) Bright field microscopy showed the morphological disparity among NSCs transfected with negative control shRNA (NC), shTtyh1, and shTtyh1+ NFATc3 (rescue group), respectively. After 48 or 72 h of transfection, the results showed that overexpression of NFATc3 partially rescued the phenotypic changes caused by knockdown of Ttyh1. (D) RT-qPCR analysis showed that when Ttyh1 was knockdown, Ccnd1 was significantly increased, and p21 was slightly increased too. When NFATc3 was overexpressed at the same time, p21 increased significantly, whereas Ccnd1 showed a down-regulated trend. Scale bar = 100 μm in C. n ≥ 4 for all experiments. Data are expressed as mean ± SEM. Statistical significance was calculated using an unpaired, two-tailed Student’s t -test. (∗∗∗∗) p < 0.0001, (∗∗∗) p < 0.001, (∗∗) p < 0.01, (∗) p < 0.05.

Article Snippet: Membranes were blocked using 5% skim milk at room temperature for 1 h, and incubated at 4°C overnight with the addition of the primary antibodies of β-actin (1:10,000, Abcam), Ttyh1 (1:400, self-prepared , STIM1 (1:400, Proteintech, Chicago, IL, United States), Orai1 (1:400, Proteintech), NFATc3 (1:500, Proteintech), CALM (1:1,000, Abcam), CaMKII (1:1,000, Abcam), Cyclin D1 (1:5,000, Proteintech), p21 (1:1,000, Abcam).

Techniques: Quantitative RT-PCR, Expressing, Control, RNA Expression, Western Blot, Microscopy, Transfection, Negative Control, shRNA, Over Expression, Knockdown, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Transmembrane Protein Ttyh1 Maintains the Quiescence of Neural Stem Cells Through Ca 2+ /NFATc3 Signaling

doi: 10.3389/fcell.2021.779373

Figure Lengend Snippet:

Article Snippet: Membranes were blocked using 5% skim milk at room temperature for 1 h, and incubated at 4°C overnight with the addition of the primary antibodies of β-actin (1:10,000, Abcam), Ttyh1 (1:400, self-prepared , STIM1 (1:400, Proteintech, Chicago, IL, United States), Orai1 (1:400, Proteintech), NFATc3 (1:500, Proteintech), CALM (1:1,000, Abcam), CaMKII (1:1,000, Abcam), Cyclin D1 (1:5,000, Proteintech), p21 (1:1,000, Abcam).

Techniques: Sequencing

Journal: Circulation Research

Article Title: STIM1 Restores Coronary Endothelial Function in Type 1 Diabetic Mice

doi: 10.1161/circresaha.112.275743

Figure Lengend Snippet: Figure 4. STIM1 overexpression restores the attenuated increase in [Ca2+]cyt due to Ca2+ release/leakage from the ER during CPA treatment in MCECs isolated from diabetic mice. A, Construction of STIM1-positive mutant with the Tie2 promoter in an adenoviral vector. B, STIM1 adenovirus (Adv) infection in HCECs upregulates STIM1 protein concentration determined by Western blot. C, STIM1 protein expression level in MCECs after Adv infection. Representative images showing STIM1 protein expression level determined by immunofluorescence in control MCECs infected with control-Adv (Cont EC–Cont Adv), diabetic MCECs infected with control-Adv (Dia EC–Cont Adv), and diabetic MCECs infected with STIM1 Adv (Dia EC–STIM1 Adv). Dark dots in the cells are beads used for cell isolation. Bar=20 μm. Lower panel shows summarized data of STIM1 expression level (intensity). Cont EC–Cont Adv, n=104 cells; Dia EC–Cont Adv, n=143 cells; Dia EC–STIM1 Adv, n=151 cells. Data are mean±SEM. *P<0.05 versus Cont EC–Cont Adv. #P<0.05 versus Dia EC–Cont Adv. D, STIM1 overexpression in MCECs isolated from diabetic mice significantly increased the rise in [Ca2+]cyt due to Ca2+ leakage but not by SOCE toward the level of control MCECs. Summarized data of the rise in [Ca2+]cyt due to Ca2+ release/leakage from the ER (1st ΔF/F0 and 1st area under the curve [AUC]) and SOCE (2nd ΔF/F0 and 2nd AUC). Control ECs infected with control Adv (Cont EC–Cont Adv, open bars), n=18; diabetic ECs infected with control Adv (Dia EC–Cont Adv, solid bars), n=22; and diabetic ECs infected with STIM1 Adv (Dia EC–STIM1 Adv, hatched bars), n=25. Data are mean±SEM. *P<0.05 versus Cont EC–Cont Adv. #P<0.05 versus Dia EC–Cont Adv. ANOVA was performed to test the statistical difference between the groups.

Article Snippet: Anti-SERCA1/2/3, anti-SERCA3, anti-tubulin, anti-actin antibodies, and STIM1 siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-STIM1 and STIM2 antibodies were from Sigma-Aldrich (St Louis, MO, USA).

Techniques: Over Expression, Isolation, Mutagenesis, Plasmid Preparation, Infection, Protein Concentration, Western Blot, Expressing, Immunofluorescence, Control, Cell Isolation

Journal: Circulation Research

Article Title: STIM1 Restores Coronary Endothelial Function in Type 1 Diabetic Mice

doi: 10.1161/circresaha.112.275743

Figure Lengend Snippet: Figure 5. STIM1 overexpression increases the [Ca2+]ER in MCECs isolated from diabetic mice. A, Typical record of the change in [Ca2+]ER in coronary ECs and the parameters used for the statistical analysis. B, Averaged record of the change in [Ca2+]ER in control MCECs infected with control-Adv (Cont EC–Cont Adv, black tracing), diabetic MCECs infected with control-Adv (Dia EC–Cont Adv, red tracing), and diabetic MCECs infected with STIM1 Adv (Dia EC–STIM1 Adv, blue tracing). C, Summarized data of resting level of [Ca2+]ER, ΔPeak, and the area under the curve (AUC; the change in [Ca2+]ER after CPA treatment). Cont EC–Cont Adv (open bars), n=25; Dia EC–Cont Adv (solid bars), n=21; and Dia EC–STIM1 Adv (hatched bars), n=30. Data are mean±SEM. *P<0.05 versus Cont EC–Cont Adv. #P<0.05 versus Dia EC–Cont Adv. ANOVA was performed to test the statistical difference between the groups.

Article Snippet: Anti-SERCA1/2/3, anti-SERCA3, anti-tubulin, anti-actin antibodies, and STIM1 siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-STIM1 and STIM2 antibodies were from Sigma-Aldrich (St Louis, MO, USA).

Techniques: Over Expression, Isolation, Control, Infection

Journal: Circulation Research

Article Title: STIM1 Restores Coronary Endothelial Function in Type 1 Diabetic Mice

doi: 10.1161/circresaha.112.275743

Figure Lengend Snippet: Figure 6. Overexpression of STIM1 restores CPA- and ACh-induced relaxation in diabetic CAs and increases nitric oxide (NO) production in diabetic MCECs. A, After precontraction of the CAs, CPA-induced (10 μmol/L) vascular relaxation was observed. Relaxation was calculated versus the magnitude of the contraction induced by PGF2α and described as percentage. Control CAs infected with control Adv (Cont CA–Cont Adv, open bar), n=4; diabetic CAs infected with control Adv (Dia CA–Cont Adv, solid bar), n=5; diabetic CAs infected with STIM1 Adv (Dia CA–STIM1 Adv, hatched bar), n=3. Data are mean±SEM. *P<0.05 versus Cont CA– Cont Adv. #P<0.05 versus Dia CA–Cont Adv. B, Endothelium-dependent relaxation was determined by ACh-induced relaxation. After preconstruction of CAs, ACh was administrated with a dose-dependent manner. Cont CA–Cont Adv (open circles), n=4; Dia CA–Cont Adv (solid circles), n=5; Dia CA–STIM1 Adv (solid triangles), n=4. Data are mean±SEM. *P<0.05 versus Cont CA–Cont Adv. #P<0.05 versus Dia CA–Cont Adv. C, Endothelium-independent relaxation was determined by SNP-induced relaxation. After preconstruction of CAs, SNP was administrated with a dose-dependent manner. Dia CA–Cont Adv (solid circles), n=3; Dia CA–STIM1 Adv (solid triangles), n=3. Data are mean±SEM. D, Resting level of DAF intensity was obtained from the average intensity of first 2 to 4 minutes during Ca2+ PSS perfusion. Cont EC–Cont Adv (open bar), n=44; Dia EC–Cont Adv (solid bar), n=49; Dia EC–STIM1 Adv (hatched bar), n=44. Data are mean±SEM. *P<0.05 versus Cont EC–Cont Adv. #P<0.05 versus Dia EC–Cont Adv. E, NO production due to Ca2+ release/leakage from the ER during CPA treatment in coronary ECs. Left graph shows a typical record of DAF-FM intensity change indicated as F/F0. The slope between time 15 (t15) and time 24 (t24) (total 9 minutes) was calculated (gray line) and used as an indication of NO production in response to CPA [d(F/F0)/dt]. Right panel shows the summarized data of d(F/F0)/dt during CPA treatment (t15-t24). Cont EC–Cont Adv (open bar), n=44; Dia EC–Cont Adv (solid bar), n=49; Dia EC–STIM1 Adv (hatched bar), n=44. Data are mean±SEM. *P<0.05 versus Cont EC–Cont Adv. #P<0.05 versus Dia EC–Cont Adv. ANOVA was performed to test the statistical difference between the groups.

Article Snippet: Anti-SERCA1/2/3, anti-SERCA3, anti-tubulin, anti-actin antibodies, and STIM1 siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-STIM1 and STIM2 antibodies were from Sigma-Aldrich (St Louis, MO, USA).

Techniques: Over Expression, Control, Infection

Journal: Circulation Research

Article Title: STIM1 Restores Coronary Endothelial Function in Type 1 Diabetic Mice

doi: 10.1161/circresaha.112.275743

Figure Lengend Snippet: Figure 7. STIM1 downregulation attenuates the rise in [Ca2+]cyt due to Ca2+ release/leakage from the ER during CPA treatment and decreases [Ca2+]ER in coronary ECs. A, STIM1 siRNA transfection in HCECs downregulates STIM1 protein expression determined by Western blot. Values are mean±SEM (n=2). *P<0.05 versus control siRNA. B, Summarized data of the rise in [Ca2+]cyt due to Ca2+ release/leakage from the ER (1st ΔF/F0 and 1st area under the curve [AUC]) and SOCE (2nd ΔF/F0 and 2nd AUC). Control siRNA (open bars), n=25 cells; STIM1 siRNA (solid bars), n=27 cells. Data are mean±SEM. *P<0.05 versus control siRNA. C, Summarized data of resting level of [Ca2+]ER, ΔPeak, and the AUC (the change in [Ca2+]ER after CPA treatment). Control siRNA (open bars), n=24; STIM1 siRNA (solid bars), n=30. Data are mean±SEM. *P<0.05 versus control siRNA.

Article Snippet: Anti-SERCA1/2/3, anti-SERCA3, anti-tubulin, anti-actin antibodies, and STIM1 siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-STIM1 and STIM2 antibodies were from Sigma-Aldrich (St Louis, MO, USA).

Techniques: Transfection, Expressing, Western Blot, Control

Journal: Circulation Research

Article Title: STIM1 Restores Coronary Endothelial Function in Type 1 Diabetic Mice

doi: 10.1161/circresaha.112.275743

Figure Lengend Snippet: Figure 8. High glucose (HG) and free fatty acid (HF) treatment downregulate protein expression of STIM1, whereas only HF decreases mRNA level of STIM1, in mouse coronary ECs. A and B, STIM1 protein (A, n=5 in each group) and mRNA (B, n=6 in each group) levels were measured in ECs treated with NG (open bars) or HG (solid bars) for 48 hours. Data are mean±SEM. *P<0.05 versus NG. C and D, STIM1 protein (A, n=3 in each group) and mRNA (B, n=6 in each group) levels were measured in ECs treated with vehicle (NF, open bars) or HF (solid bars) for 24 hours. Data are mean±SEM. *P<0.05 versus NF.

Article Snippet: Anti-SERCA1/2/3, anti-SERCA3, anti-tubulin, anti-actin antibodies, and STIM1 siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-STIM1 and STIM2 antibodies were from Sigma-Aldrich (St Louis, MO, USA).

Techniques: Expressing

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Photostimulation was applied at 470 nm (4.0 mW/cm 2 ). Data were shown as mean ± sem. Scale bar, 5 µm. a Domain architecture of the human STIM1. SP, signal peptide; EF-SAM, EF-hand and sterile alpha-motif; TM, transmembrane domain; CC1, coiled-coil domain 1; SOAR, STIM-Orai activating region; P/S, proline/serine-rich region; TRIP, the S/TxIP microtubule-binding motif; PB, polybasic tail. b Schematic of STIM1–ORAI1 coupling at the ER–PM junction that mediates store-operated Ca 2+ entry. c – e Use of the iLID-sspB optical dimerizer to trigger STIM1ct activation and Ca 2+ influx through endogenous ORAI channels. c Schematic of the design. iLID or sspB was fused to the N-terminus of STIM1ct at residue 233. d Confocal images showing photoswitchable Ca 2+ influx in HeLa cells co-transfected with a red Ca 2+ sensor (R-GECO 1.2) and the iLID/sspB fused STIM1ct chimeras. Cells were exposed to two repeated dark-light cycles. e Quantitative analysis of Ca 2+ signals in response to repeated photostimulation ( n = 40 cells from three independent experiments). The half-lives ( t 1/2 ) of on and off kinetics were fitted with one phase exponential decay (“±” means 95% confidence interval). f – h Use of the CRY2-CIBN optical dimerizer to photo-activate STIM1ct and Ca 2+ influx. f Schematic of the design. CRY2 was used to photo-crosslink CIBN-STIM1ct and trigger STIM1ct activation to induce Ca 2+ entry. g Confocal images showing light-induced co-localization of mCherry (mCh)-tagged CIBN-STIM1ct with YFP-ORAI1 in HeLa cells. h Reversible Ca 2+ responses monitored by R-GECO 1.2 ( n = 30 cells). Blue bar, photostimulation at 470 nm with a power density of 4 mW/cm 2 . i – k ER-tethered CRY2-STIM1ct mimics STIM1 puncta formation at ER–PM junctions to evoke localized Ca 2+ influx. i Schematic of the design. j Confocal images illustrating light-induced clustering of ER-resident CRY2-STIM1ct at the footprint of HeLa cells. Enlarged views of the boxed regions were shown on the right. k Cytosolic Ca 2+ signals reported by R-GECO1.2 in HeLa cells subjected to two repeated dark-light cycles ( n = 30).

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Binding Assay, Activation Assay, Transfection

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of a split STIM1 molecule (at residue 342) to monitor CC1–SOAR interaction in trans at real time. CC1–SOAR maintains STIM1ct in an inactive configuration at rest. As a result, Part II (mCh-STIM1 342–685 ) tightly docks to the ER-resident Part I (STIM1 1–342 -YFP) when the store remains full. Upon store depletion, structural changes propagate toward the CC1 region to weaken its association with SOAR, thereby leading to the cytosolic dispersion of Part II as shown in panel b . b Confocal images showing the distribution of split STIM1 molecules (green, STIM1 1–342 -YFP; red, mCh-STIM1 343–685 ) before and after thapsigargin (TG)-induced store depletion in HeLa cells. c Schematic illustrating the design of a LOV2-SOAR (STIM1 336–486 ) chimera to mimic the CC1–SOAR interaction that locks STIM1ct in an inactive state. CC1 is replaced by LOV2 (light-oxygen-voltage domain 2) to tightly cage SOAR in the dark. Upon blue light stimulation, the Jα helix becomes disordered to uncage SOAR, thereby restoring its activity to engage and gate ORAI channels. If the ER-resident Part I (STIM1 1–342 -YFP) and PM-embedded ORAI1 are co-expressed, LOV2-SOAR can be used to determine the relative binding strength of SOAR toward ER-anchored CC1 or PM-resident ORAI1 channels. d Light-inducible cytosol-to-ER translocation of mCh-LOV2-SOAR in HEK293 cells co-transfected with Part I as shown in panel c . e Quantification of cytosolic mCherry signals (images in panel d ) following three repeated light-dark cycles ( n = 18 cells). f – h Comparison of the relative strength of SOAR-CC1 and SOAR-ORAI1 interactions. f Top: Light-induced cytosol-to-PM translocation of mCh-LOV2-SOAR (gray) observed in HEK293 cells co-transfected with YFP-ORAI1 (green). Bottom: Confocal images of HEK293 cells co-expressing mCh-LOV2-SOAR (gray), YFP-ORAI (green) and STIM1 1–342 -CFP (cyan). In the dark, mCh-LOV2-SOAR was evenly distributed in the cytosol. Upon photostimulation, mCh-LOV2-SOAR preferred to translocate toward ER membrane but not to PM. g The fluorescence intensities (YFP, green; mCh, red; CFP, cyan) across the dashed line were plotted to evaluate the degree of colocalization. h Light-induced Ca 2+ response curves (quantified by GCaMP6s) in HEK293 cells transfected with LOV2-SOAR (red), LOV2-SOAR + ORAI1 (green) or LOV2-SOAR + Part I (STIM1 1–342 ; blue). n = 30 cells.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Activity Assay, Binding Assay, Translocation Assay, Transfection, Expressing, Fluorescence

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of an optogenetic clustering assay to examine real-time protein–protein interactions in living cells. b Summary of mCh-tagged baits and YFP-tagged preys used to map critical domains in STIM1ct that dictate STIM1 oligomerization. c Representative confocal images showing the intracellular distribution of the bait (mCh-CRY2-STIM1 233–448 ) and two different preys (P1—top panel, YFP-STIM1 233–658 ; P3—bottom panel, YFP-STIM1 233–342 ) before and after blue light stimulation in HeLa cells. d Time courses showing the kinetics of light-induced clustering (F cluster /F neighbor ) of the bait and its co-clustering with P1 (blue), but not with P3 (green), as seen in panel c . n = 18 cells. ( e ) Quantification of the degrees of light-inducible co-clustering for the five indicated preys in HeLa cells co-transfected with the bait. n = 18 cells from three independent experiments. f Summary of the optogenetic co-clustering assay results for the bait–prey combinations shown in panel b . “+” means co-clustering notably observed after photo-illumination; “−” means no appreciable cluster formation before and after blue light stimulation. g – i Light inducible co-clustering to dissect the STIM1 luminal EF-SAM domain. g Schematic showing the design of bait–prey constructs. h – i Representative confocal images of HeLa cells co-expressing ( h ) mCh-CRY2-EF (B4, STIM1 32–128, EF-hand ; red) with YFP-EF (P6, green), or ( i ) mCh-CRY2-SAM (B5, STIM1 128–200, SAM ; red) with YFP-SAM (P7; red) under dark (left) and blue light (right). The selected regions (dashed boxes) were enlarged to aid visualization. Scale bar 5 µm.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Transfection, Construct, Expressing

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of the high-throughput screening pipeline. The cytosol-to-PM translocation and Ca 2+ influx (GCaMP6s as readout) were used as two readouts. b Sequence alignment of human SOAR1 and SOAR2 domains and the 3D structure of SOAR1 (PDB entry: 3TEQ). Key residues at the interdimer interface or involved in ORAI1-binding were indicated by dots and triangles, respectively. Selected key residues were highlighted in the 3D structure. c Quantification of Ca 2+ responses (GCaMP6s) and PM translocation (mCherry signals) of selected CRY2-STIM1ct mutants before (dark dots) and after (blue dots) photostimulation. HeLa-GCaMP6s stable cells were co-transfected with each of the indicated mCh-CRY2-STIM1ct mutants and ORAI1-CFP. d Time courses showing the kinetics of light-induced Ca 2+ influx for WT and the indicated mCh-CRT2-STIM1ct variants. n = 60 cells. e – g Representative confocal images ( e ) and quantification of intracellular Ca 2+ signals, n = 60 cells. Box-whisker plots indicated the median, and the interquartile range with 5–95 percentile distribution. f , as well light-induced PM translocation, n = 8 cells ( g ), in HeLa cells expressing WT or the indicated mCh-CRY2-STIM1ct mutants. h Representative confocal images of HEK293 S1-KO cells expressing the GFP-tagged full-length STIM1-T393F mutant before and after TG-induced store depletion. i SOCE monitored by R-GECO1.2 in HEK293 S1-KO cells expressing GFP-STIM1 WT or the mutant T393F. n = 90 cells. j Summary of the degrees of Ca 2+ influx and PM translocation of cancer-associated mutations found in the SOAR domains of STIM1. HeLa cells were transfected with the indicated mCh-CRT2-STIM1ct mutants. Gain-of-function (H395Y and R424W; red) and loss-of-function (L402R, R426L/C, R429C; green) mutations were both identified. n = 60 cells.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: High Throughput Screening Assay, Translocation Assay, Sequencing, Binding Assay, Transfection, Whisker Assay, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Diagram of the STIM1 C-terminal fragment (residues 443–685) that contains both the S/TxIP EB1-binding motif and the positively charged polybasic tail (PB) that interacts with PM-resident PIPs. Mutations used in this study to perturb STIM1ct-target interactions were highlighted in red. b , c Schematic ( b ) and representative confocal images ( c ) showing light-inducible bimodal distribution (tracking of MT plus ends or cytosol-to-PM translocation) of mCh-CRY2-STIM1 443–685 in COS-7 cells. d Time courses of light-triggered MT plus-end tracking (green) and PM translocation (blue) of mCh-CRY2-STIM1 443–685 . Upon blue light illumination, cytosolic mCherry signals rapidly reduced ( t 1/2 = 18.2 ± 5.0 s), accompanied with the increase of MT tip tracking ( t 1/2 = 21.5 ± 7.3 s) or PM decoration ( t 1/2 = 27.6 ± 6.2 s). n = 12 cells. e , f Representative confocal images ( e ) and quantifications of MT plus-end tracking ( f ) of mCh-CRY2-STIM1ct variants (top, WT; bottom, P645N in the context of STIM1 443–670 ) in COS-7 cells co-transfected with EB1-GFP (green). The bar graph showed the averaged values of MT tip-to-cytosol intensity ratio under dark and lit conditions. n = 16 cells from three independent experiments. g , h Representative confocal images in HeLa cells ( g ) and quantification of the cytosol-to-PM translocation ( h ) of the indicated CRY2-PB variants before and after blue light illumination. n = 16 cells from three independent experiments. i – k Schematic showing light inducible ER-MT interactions in COS-7 cells cotransfected ER-anchored mCh-CRY2-STIM1 443–670 (red) with ( j ) EB1-GFP (green) or ( k ) GFP-tubulin (green). j Representative confocal images showing ER morphology change following blue light illumination. The mCherry and GFP fluorescent intensities across the dashed line were plotted next to the images to indicate the degree of signal overlaps. k Confocal images showing the clustering of ER-resident mCh-CRY2-STIM1 443–670 along with GFP-tubulin (green) upon blue light illumination. The surface profiles of a selected area (oval) were presented to aid the visualization of subcellular distribution. l , m Light-inducible assembly of ER-plasma membrane contact sites (MCSs) mediated by ER-resident mCh-CRY2-PB (STIM1 671–685 ). l Schematic of the design. m Representative confocal images of the footprint of HeLa cells transfected with mCh-CRY2-PB variants before and after blue light illumination.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Binding Assay, Translocation Assay, Transfection