Journal: Biochimica et biophysica acta

Article Title: Epoxyeicosatrienoic acids act through TRPV4-TRPC1-KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries.

doi: 10.1016/j.bbadis.2014.12.010

Figure Lengend Snippet: Fig. 1. Co-immunoprecipitation of TRPV4, TRPC1 and KCa1.1(α) in the lysates of human LIMA VSMCs. A and B, TRPC1 with KCa1.1(α). C and D, TRPC1 with TRPV4. E and F, TRPV4 with KCa1.1(α). The data are representative of 3 to 4 experiments. The pulling antibody and the blotting antibody were indicated (+). Control immunoprecipitation (−) was performed using the preimmune IgG. IP, immunoprecipitation; Blot, immunoblot.

Article Snippet: The primary rabbit antibodies against KCa1.1(α) (APC-021), TRPC1 (ACC-010), and TRPV4 (ACC-034) were from Alomone Labs. T1E3 was raised by us from rabbits.

Techniques: Immunoprecipitation, Control, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Epoxyeicosatrienoic acids act through TRPV4-TRPC1-KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries.

doi: 10.1016/j.bbadis.2014.12.010

Figure Lengend Snippet: Fig. 5. Membrane potential changes in HEK293 cells that over-expressed with TRPV4, TRPC1 and KCa1.1. A and B, representative traces illustrating the hyperpolarizing responses to 300 nmol/L 11,12-EET (A), 5 μmol/L 4a-PDD (B) and 100 nmol/L GSK1016790A (C). D–F, summary of data showing the maximal hyperpolarization to 300 nmol/L 11,12-EET (D), 5 μmol/L 4a- PDD (E) and 100 nmol/L GSK1016790A (F) under different conditions. C1mut-pore, TRPC1 were replaced by a TRPC1 pore-dead mutant C1mut-pore. Values are means ± SE (n = 5 to 9). **, P b 0.01 compared to the controls (non-transfected); ##, P b 0.01 compared to the transfected cells without inhibitors; $$, P b 0.01 compared to preimmune IgG.

Article Snippet: The primary rabbit antibodies against KCa1.1(α) (APC-021), TRPC1 (ACC-010), and TRPV4 (ACC-034) were from Alomone Labs. T1E3 was raised by us from rabbits.

Techniques: Membrane, Mutagenesis, Transfection

Journal: Biochimica et biophysica acta

Article Title: Epoxyeicosatrienoic acids act through TRPV4-TRPC1-KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries.

doi: 10.1016/j.bbadis.2014.12.010

Figure Lengend Snippet: Fig. 4. Requirement of TRPC1 for TRPV4 and KCa1.1 interaction. A–D, co-immunoprecipitation of TRPV4 and KCa1.1(α) in the lysates of HEK293 cells. A and B, HEK293 cells that over- expressed with TRPV4, TRPC1 and KCa1.1. C and D, HEK293 cells that over-expressed with TRPV4 and KCa1.1 but without TRPC1. The right lanes in C and D were immunoblots using cell lysates without immunoprecipitation. The pulling antibody and the blotting antibody were indicated (+). Control immunoprecipitation (−) was performed using the preimmune IgG. IP, immunoprecipitation; Blot, immunoblot. n = 3 to 4 experiments. E–F, the maximal hyperpolarization in response to 300 nmol/L 11,12-EET (E) and 5 μmol/L 4α-PDD (F) in HEK293 cells that were transfected with indicated constructs. C1 stands for TRPC1. V4 stands for TRPV4. Values are means ± SE (n = 5 to 9). **P b 0.01 compared to the controls (non-transfected), ##P b 0.01 compared to C1 + V4 + KCa1.1.

Article Snippet: The primary rabbit antibodies against KCa1.1(α) (APC-021), TRPC1 (ACC-010), and TRPV4 (ACC-034) were from Alomone Labs. T1E3 was raised by us from rabbits.

Techniques: Immunoprecipitation, Western Blot, Control, Transfection, Construct

Journal: Cell Research

Article Title: Direct control of store-operated calcium channels by ultrafast laser

doi: 10.1038/s41422-020-00463-9

Figure Lengend Snippet: a Ca 2+ influx intensity of cells treated with 2-APB ( n = 132, 4 trials; n = 129 cells, 5 trials), SKF-96365 ( n = 60, 4 trials; n = 108 cells, 6 trials), YM-58483 ( n = 57, 3 trials; n = 57 cells, 3 trials), and GSK7975A ( n = 60 cells, 3 trials; n = 60 cells, 3 trials) at two different concentrations for a duration of 70 s after the femtoSOC laser illumination. b Add-back test showing Ca 2+ influx suppressed by adding YM-58483 before the femtoSOC laser illumination. Red arrow, timing of the femtoSOC laser illumination. Right, statistics of the measured Ca 2+ influx peak level with or without YM-58483 ( n = 13 cells and n = 17 cells in the control and YM-58483 groups, respectively, P = 0.0061). c Add-back test showing Ca 2+ influx suppressed by adding YM-58483 30 s after the femtoSOC laser illumination. Red arrow, timing of the femtoSOC laser illumination. Orange arrow, timing of the YM-58483 addition. Right, statistics of the Ca 2+ influx decay time defined by the duration of the decay to reach the half maximum of the peak value ( n = 30 cells and n = 24 cells in the control and YM-58483 groups, respectively, P < 0.0001). d Ca 2+ influx probability of cells with Orai1 knockdown ( n = 144 cells, P = 0.0057). Inset, western blot analysis of Orai1 knockdown. e Ca 2+ influx probability of cells with Orai1 overexpression ( n = 135 cells, P = 0.001). Inset, western blot analysis of Orai1 overexpression. f Ca 2+ influx intensity in Orai1/2/3-KO cells compared with that in control cells or that in Orai1/2/3-KO cells with Orai1-mCherry introduced. g No Ca 2+ influx in Orai1-KO cells. h Electrophysiological current of Ca 2+ influx measured by the patch-clamp technique. i Confirmed absence of Zn 2+ and Mg 2+ influx. j Ca 2+ influx probability of cells with TRPC1 knockdown by shRNA ( n = 114 cells, 5 trials, P = 0.3369). Inset, western blot analysis of TRPC1 knockdown. * P < 0.05. ** P < 0.01. *** P < 0.001. **** P < 0.0001. NS, no significant difference. Scale bar, 10 μm.

Article Snippet: Antibodies for immunoblotting were as follows: Orai1 rabbit polyclonal antibody (13130-1-AP, Proteintech, 1:100 dilution), rabbit anti-ORAI1 (O8264, Sigma, 1:1000 dilution), TRPC1 rabbit polyclonal antibody (19482-1-AP, Proteintech, 1:500 dilution), GAPDH mouse monoclonal antibody (60004-1-Ig, Proteintech, 1:10,000 dilution), Peroxidase AffiniPure goat anti-mouse IgG (H + L) (115-035-003, Jackson ImmunoResearch, 1:10,000 dilution), Peroxidase AffiniPure goat anti-rabbit IgG (H + L) (111-035-003, 1:10,000 dilution, Jackson ImmunoResearch).

Techniques: Control, Knockdown, Western Blot, Over Expression, Patch Clamp, shRNA

Journal: Journal of Biological Chemistry

Article Title: Inositol 1,4,5-Trisphosphate (IP3) Receptor Up-regulation in Hypertension Is Associated with Sensitization of Ca2+ Release and Vascular Smooth Muscle Contractility

doi: 10.1074/jbc.m113.496802

Figure Lengend Snippet: FIGURE 2. Screening of Ca2-handling proteins reveals coupled up-regu- lationofthevascularIP3RandLTCCinexperimentalmodelsofhyperten- sion. A, immunoblot analysis of IP3R1, 1C, TRPC1, TRPC4, Orai1, and STIM1 in lysates from mouse MA. MA lysate pooled from three SAL or AHT mice was added to each lane. Increased expression levels of IP3R1, 1C, and TRPC4 are detected in MA of AHT mice, whereas levels of TRPC1, Orai1, and STIM1 were similar. B, agarose gel analysis of products obtained after PCRs for IP3R1 (274 bp), 1C (185 bp), and TRPC4 (226 bp) using specific primers. -Actin (152 bp) and vWF (178 bp) were used as VSM and endothelial cell markers, respec- tively.IP3R,1C,and-actinweredetectedbothinintactMAandisolatedVSM cells as expected. In contrast, TRPC4 and vWF were detected in arteries but not isolated VSM cells, implying expression only in endothelium. C, immuno- blots comparing the expression of IP3R, 1C, PMCA, and -actin (as a loading control) between MA from normotensive Wistar Kyoto rats (WKY) and SHR and between MA from sham-operated and aortic-banded (Band) hyperten- sive rats. In each case, lysates were pooled from arteries of three or four rats. Only the IP3R and 1C proteins are up-regulated in MA from hypertensive SHR and aortic-banded rats. Blots are representative of three experiments.

Article Snippet: Forty g of protein was loaded per well, and expression of Ca2 -handling proteins was detected using specific antibodies against IP3R1 (1:1000; Millipore), 1C (1:1000; Millipore), TRPC1 (1:200; Santa Cruz Biotechnology), TRPC4 (1:200;NovusBiologicals), STIM1 (1:1000; Cell SignalingTechnology), Orai1 (1:1000; Sigma), and PMCA1 (1:500; Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Agarose Gel Electrophoresis, Isolation, Control

Journal: Journal of Biological Chemistry

Article Title: Inositol 1,4,5-Trisphosphate (IP3) Receptor Up-regulation in Hypertension Is Associated with Sensitization of Ca2+ Release and Vascular Smooth Muscle Contractility

doi: 10.1074/jbc.m113.496802

Figure Lengend Snippet: FIGURE 3. Depolarization of A7r5 cells for 24 h replicates the induction of IP3R and 1C observed in VSM cells of hypertensive animals in vivo. A–E, immunoblotanalysesofIP3R,PMCA,and1C.ProteinswereisolatedfromA7r5cellsandseparatedonSDS-PAGE,andtheblotswereprobedfortheappropriate antigens as indicated. A, IP3R1 expression is increased in A7r5 cells exposed to K20, K40, or K60 for 24 h but not in cells treated with NaCl or sucrose (Sucr) as osmolar controls. Depolarization-induced IP3R up-regulation is lost in the presence of the LTCC blocker, Nif (10 M). Expression levels of PMCA are unchanged. B, the expression level of 1C is significantly increased in A7r5 cells incubated with K20 or K40 for 24 h but not in cells exposed to sucrose or 10 M Nif (n 3). C, expression levels of TRPC1, STIM1, and -actin are not altered in A7r5 cells depolarized for 24 h by 20K. D, right blot shows that IP3R1 up-regulates in A7r5 cells exposed to 20K or treated with Thaps (1 M) for 24 h. The up-regulation of IP3R1 by K20 is blocked by 10 g/ml CHX or 10 M ACD. Blots are representative of at least three independent experiments. Smooth muscle-specific -actin was used as a loading control. E, relative immunodensity of the IP3R1 after normal- ization to -actin. Cell depolarization with K20 or treatment with Thaps (1 M) for 24 h increases IP3R1 expression, and this response is lost in the presence Nif (10 M), CHX (10 g/ml), ACD (10 M), or CsA (10 M) (n 3–21; *, p 0.05; **, p 0.01). F, transcriptional up-regulation of IP3R1, IP3R3, and 1C in A7r5 cells depolarized by K20 for 24 h. Real-time PCR plots represent relative quantification. G, nuclear translocation of NFATc1 in A7r5 cells. Cells were transfected with plasmids expressing EGFP-NFATc1 and imaged using a confocal laser-scanning microscopy. At rest (Con), NFATc1 showed a basal cytoplasmic localization, but treatment with Thaps (1 M) or K20 alone or in the presence of CHX (10 g/ml), ACD (10 M), or CsA (10 M) promotes its nuclear translocation. In contrast, incubation of cells with 10 M Nif or CsA prevents K20-induced nuclear translocation of NFATc1. Data are representative of four independent experiments. Scale bar, 20 m. Error bars, S.E.

Article Snippet: Forty g of protein was loaded per well, and expression of Ca2 -handling proteins was detected using specific antibodies against IP3R1 (1:1000; Millipore), 1C (1:1000; Millipore), TRPC1 (1:200; Santa Cruz Biotechnology), TRPC4 (1:200;NovusBiologicals), STIM1 (1:1000; Cell SignalingTechnology), Orai1 (1:1000; Sigma), and PMCA1 (1:500; Santa Cruz Biotechnology).

Techniques: In Vivo, Expressing, Incubation, Control, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Translocation Assay, Transfection, Confocal Laser Scanning Microscopy

Journal: Journal of Biological Chemistry

Article Title: Ca2+ Entry via TRPC Channels Is Necessary for Thrombin-induced NF-κB Activation in Endothelial Cells through AMP-activated Protein Kinase and Protein Kinase Cδ

doi: 10.1074/jbc.m803984200

Figure Lengend Snippet: FIGURE 1. Store-operated Ca2 influx in endothelial cells is required for thrombin-induced NF-B activation. A, HPAECs were transfected with either 100 nM Sc-siRNA or TRPC1 siRNA. At 72 h after transfection, cells were lysed and immunoblotted with anti-TRPC1 polyclonal Ab (top). The mem- brane was stripped and blotted with anti--actin mAb (bottom). B, control HPAECs and HPAECs transfected with Sc-siRNA or TRPC1 siRNA were used to measure thrombin-induced store Ca2 release and Ca2 release-activated Ca2 influx (see details under “Experimental Procedures”). The results were compared with nontransfected control cells. C, control HPAECs and HPAECs transfected with Sc-siRNA or TRPC1 siRNA were exposed to thrombin (50 nM) or TNF- (1000 units/ml) for the indicated time periods. EMSA was performed as described under “Experimental Procedures.” Experiments were repeated 3–4 times with similar results. D, control HPAECs and HPAECs transfected withTRPC1siRNAasdescribedunder“ExperimentalProcedures.”At72hafter transfection, cells were exposed to thrombin for different time intervals. After thrombin treatment, nuclear and cytoplasmic fractions prepared were used for immunoblotting. The cytosolic (Cy) fractions were immunoblotted with anti- IB antibody (top panel). The membrane was stripped and probed for cytosolic marker with anti- actin monoclonal antibody (second panel). The nuclear frac- tions (Nu) were immunoblotted with anti-p65 Ab (third panel). The membrane was stripped and probed with anti-MCM3 antibody (nuclear marker).

Article Snippet: Firefly (Photinus pyralis) and sea pansy (R. reniformis) luciferase activitywere assayed by the dual luciferase reagent assay system (19). siRNA Transfection—HPAECs grown to 70% confluence on gelatin-coated culture dishes were transfected with either 100 nM AMPK 1 siRNA, TRPC1 siRNA, or Sc-siRNA using Santa Cruz Biotechnology transfection reagents.

Techniques: Activation Assay, Transfection, Control, Western Blot, Membrane, Marker

Journal: Journal of Biological Chemistry

Article Title: Ca2+ Entry via TRPC Channels Is Necessary for Thrombin-induced NF-κB Activation in Endothelial Cells through AMP-activated Protein Kinase and Protein Kinase Cδ

doi: 10.1074/jbc.m803984200

Figure Lengend Snippet: FIGURE 5. Thrombin-induced AMPK activation phosphorylates p38 MAP kinase,p65/RelA,andPKCinendothelialcells.A,HPAECswereincubated in the presence and absence of compound C (40 M) for 30 min and then stimulated with thrombin (50 nM) for different time intervals. After thrombin treatment, cells were lysed and immunoblotted with anti-phospho-p38 MAP kinase mAb. The membrane was stripped and blotted with anti-p38 MAP kinase Ab for total p38 MAP kinase. B, HPAECs transfected with the Sc-siRNA or AMPK siRNA were challenged with thrombin (50 nM) for different time intervals. After thrombin treatment, cells were lysed and immunoblotted with anti-phospho-p38 MAP kinase mAb and p38 MAP kinase Ab. The exper- iment was repeated three times. The results from representative experiments are shown. C, HPAECs transfected with the Sc-siRNA or AMPK siRNA were challenged with thrombin (50 nM) for 0, 10, and 30 min. After thrombin treat- ment, cells were lysed and immunoblotted with anti-phospho-Ser536-p65 mAb and anti-p65 Ab. D, control HPAECs and HPAECs transfected with TRPC1 siRNA were challenged with thrombin for different time intervals. After thrombin challenge, cells were lysed and immunoblotted with anti-phospho- PKC (Thr505) Ab. The blots were stripped and probed with anti-PKC Ab. The experiment was repeated three times. The results from representative exper- iments are shown. E, lung endothelial cells from WT and Trpc4/ mice type were challenged with thrombin (50 nM) for the indicated times. After throm- bin challenge, cells were lysed and immunoblotted with anti-phospho-PKC (Thr505) Ab. The experiment was repeated three times. F, control HPAECs and HPAECs transfected with AMPK siRNA were challenged with thrombin for different time intervals. After thrombin challenge, cells were lysed and immu- noblotted with anti-phospho-PKC (Thr505) or anti-PKC antibodies. The experiment was repeated three times. The results from representative exper- iments are shown.

Article Snippet: Firefly (Photinus pyralis) and sea pansy (R. reniformis) luciferase activitywere assayed by the dual luciferase reagent assay system (19). siRNA Transfection—HPAECs grown to 70% confluence on gelatin-coated culture dishes were transfected with either 100 nM AMPK 1 siRNA, TRPC1 siRNA, or Sc-siRNA using Santa Cruz Biotechnology transfection reagents.

Techniques: Activation Assay, Membrane, Transfection, Control

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 1. Comparison of TRPC expression levels in control and NGF-differentiated PC12 cells. A: Representative bright field images of undifferentiated(control)andNGF-differentiated(NGF)PC12cellsareshown.B:RT-PCRanalysisofTRPCexpressioninPC12cells.PCRproducts amplified using TRPC specific primer sets were separated on ethidium bromide stained agarose gels. A representative gel image is shown. C: Comparison of TRPC expression in control (C) and NGF-differentiated PC12 cells (NGF). Each experiment was repeated 7–9 times.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Comparison, Expressing, Control, Staining

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 2. Western blot analysis of TRPC protein expression in PC12 cells. A: Monoclonal anti-TRPC1 antibody (a generous gift from Dr. Leonidas Tsiokas) was used to detect the TRPC1 protein in control (C) and NGF-differentiated PC12 cells (NGF). The specificity of monoclonal anti-TRPC1 was confirmed using mouse recombinant TRPC proteins. B: TRPC5 protein expression was detected only in undifferentiated PC12 cells. C: NGF pretreatment up-regulated TRPC6 in PC12 cells. In (A)–(C), the lanes labeled mTRPC1, mTRPC5, and hTRPC6 represent recombinant proteins used as controls. ‘‘C’’ denotes control, protein extracts of untreated PC12 cells. ‘‘NGF’’ labels protein extracts isolated from PC12 cells treated with NGF for 48 h. D: Comparison of relative TRPC protein expression levels in control and NGF-differentiated PC12 cells. Each experiment was repeated 10–16 times.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Western Blot, Expressing, Control, Recombinant, Labeling, Isolation, Comparison

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 3. DN-TRPC6 facilitates NGF-induced neurite outgrowth in PC12 cells. A: Comparison of total neurite length per cell in PC12 cells electroporated with a mixture of the YFP plasmid and the pcDNA3 plasmid with or without an insert as indicated. Each group consisted of 35–312 cells. B: Comparison of total neurite length per cell inPC12 cells electroporated with GFP-tagged TRPC constructasindicated. C: Representative images demonstrating the effect of GFP-TRPC6 and DN–GFP–TRPC6 overexpression on the total neurite length per cell. In these pseudocolor images, the red color corresponds to the highest YFP fluorescence, whereas the blue color indicates the lowest YFP fluorescence.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Comparison, Plasmid Preparation, Over Expression

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 5. The effect of hyperforin on neurite outgrowth and TRPC expression in NGF-differentiated PC12 cells. A: Comparison of the total neurite length per cell in hyperforin-treated PC12 cells (n U 405–635). B, C, and D: Hyperforin-induced changes in relative TRPC channel expression in PC12 cells (n U 4 in each group). E: Representative images of gels used during the RT-PCR analysis. HF, hyperforin; Control, undifferentiated PC12 cells; NGF, NGF-differentiated PC12 cells.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Expressing, Comparison, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 4. Electrophysiological characterization of GTPgS-activated currents inTRPC5-overexpressing cells. A: Currents with TRPC5-like current–voltage relationships were observed in the round TRPC5 expressing PC12 cells (n U 8). B: Heteromeric TRPC1–TRPC5 current–voltage relationships were seen in the TRPC5- overexpressing PC12 cells exhibiting neurites (n U 8). The insets in the quadrant II show representative images of TRPC5-expressing PC12 cells, whereas the insets in the quadrant IV show sample current–voltage relationships of the homomeric TRPC5 channel or the TRPC1–TRPC5 heteromeric channel heterologously expressed in HEK cells. The solid line corresponds to the averaged current amplitudes, whereas the vertical bars represent the SEM.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Expressing

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 6. Receptor tyrosine kinase TrkA is not involved in the NGF- induced up-regulation of TRPC1 and TRPC6. A: RT-PCR analysis of TRPC1 (upper panel) and TRPC6 (lower panel) expression in NGF-treated PC12 cells in the absence or presence of 500 nM GW441756, a potent TrkA inhibitor. B: Comparisons of the relative expression levels of TRPC1 and TRPC6 (left, n U 6–7), and the total neurite length per cell (right, n U 276–290) in NGF treated PC12 cells in the absence or presence of 500 nM GW441756.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 7. IKK2 inhibitors abolished NGF-mediated increases of TRPC1 and TRPC6 channel expression in NGF-treated PC12 cells. A: Two different IKK2 inhibitors, BMS (BMS-345541, 10 mM) and IMD (IMD-0354, 1 mM), potently inhibited neurite outgrowth in NGF-differentiated PC12 cells (n U 94–383). B: RT-PCR analysis of TRPC expression in the IKK2 inhibitor treated NGF-differentiated PC12 cells (upper and middle panel). The lower panel shows the effect of the tested IKK2 inhibitors on basal TRPC1 and TRPC5 expression levels in control PC12 cells. TRPC6 expression level was very low in control PC12 cells and was not significantly altered in IKK2 inhibitor experiments (see summary in (C)). Representative gels are shown. C: The summary of RT-PCR analysis of relative TRPC expression in IKK2 inhibitor treated groups. ‘‘NGF R BMS’’ stands for NGF-differentiated PC12 cells treated with (BMS-345541, 10 mM). ‘‘NGF R IMD’’ stands for NGF-differentiated PC12 cells treated with (IMD-0354, 1 mM). ‘‘C’’ denotes undifferentiated PC12 cells. ‘‘NGF’’ denotes NGF differentiated PC12 cells. Each experiment was repeated 4–13 times.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 8. The effect of overexpression of a constitutively active mutant of IKK2 (S177E/S181E, IKK2ca) on TRPC channel expression in PC12 cells. A: IKK2ca-overexpressing undifferentiated PC12 cells exhibited longer neurites than non-transfected cells at 48 h post-electroporation time. Shownisanoverlayofthefluorescenceandcorrespondingbrightfieldimages.TheYFPfluorescenceisshowninpseudo-color.B:Comparisonofthe total neurite length per cell in pcDNA3 and IKK2ca-overexpressing undifferentiated PC12 cells (n U 231–267). C and D: The RT-PCR analysis of TRPC1 and TRPC6 expression in PC12 cells expressing either pcDNA3 or IKK2ca. E, F, and G: The relative changes in the expression of TRPC channels induced by the overexpression of IKK2ca. Each experiment was repeated 10–12 times.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Over Expression, Mutagenesis, Expressing, Transfection, Electroporation, Reverse Transcription Polymerase Chain Reaction

Journal: Brain research bulletin

Article Title: Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke.

doi: 10.1016/j.brainresbull.2024.110894

Figure Lengend Snippet: Fig. 1. Canonical transient receptor potential channel (TRPC) 1 expression is upregulated in microglia after stroke. A Quantitative polymerase chain reaction to assess the expression of Trpcs in primary cultured microglia underwent oxygen-glucose deprivation/regeneration (OGD/R) injury, expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) RNA and expressed as % relative to Gapdh (n = 6 per group). B TRPC1 protein levels in primary cultured microglia, neurons, and astrocytes were detected by Western blot assay upon OGD/R condition, each group (Normoxia or OGD/R) consisted of 3 bands from one sample. Each point represents the average intensity of 3 bands. Data are presented as percentages in comparison to the Normoxia group (n = 3). C Representative immunofluorescence staining for microglia marker, glial fibrillary acidic protein (GFAP, red) and TRPC1 (green) from the peri-infarct area of brain tissue after middle cerebral artery occlusion reperfusion. 4’,6-diamidino-2-phenylindole was used to stain the nuclei (blue). Scale bars, 100 µm. Immunofluorescence intensity of TRPC1 was quantified using ImageJ software and shown in right panel (n = 4 per group). ** P < 0.01.

Article Snippet: Microglia Trpc1 conditional knockdown (Trpc1 KD) mice were generated by mating Trpc1flox/flox (Cyagen Incorporated, Suzhou, China) and CX3C chemokine receptor 1 (Cx3cr1)CreER (Cyagen Incorporated) mice of C57BL/6 background.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Western Blot, Comparison, Immunofluorescence, Staining, Marker, Software

Journal: Brain research bulletin

Article Title: Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke.

doi: 10.1016/j.brainresbull.2024.110894

Figure Lengend Snippet: Fig. 2. Generation and characterization of conditional microglia Canonical transient receptor potential channel 1 knockdown (Trpc1 KD) mice. A Construction and breeding procedure of Trpc1 KD mice. Trpc1 KD mice possess both heterozygous Cx3cr1CreER allele and homozygous loxP-bordered Trpc1 allele. B Microglia were isolated from the cortex of Trpc1 KD mice 24 h after middle cerebral artery occlusion reperfusion (MCAO/R) via magnetic-activated cell sorting method. Knockdown efficiency of TRPC1 was confirmed by Western blot analysis, with microglia isolated from littermate Trpc1 flox/flox/Cre- mice serving as the Control group. Quan titation and statistical analysis of TRPC1 intensity were shown in the right panel, band intensity was normalized to β-actin expression. Data are presented as per centages in comparison to the Control group (n = 3 mice per group). C Body weight of Control and Trpc1 KD mice during 2–12-week development (n = 8 mice per group). D Representative immunofluorescence staining for microglia marker, Ionized calcium-binding adapter molecule 1 (Iba1, Red), astrocytic marker, glial fibrillary acidic protein (GFAP, Green), and neuron marker, neuron-specific nuclear protein (NeuN, Green) in brain section of Trpc1 KD and Control mice. 4’,6- diamidino-2-phenylindole (DAPI) was used to stain the nuclei (blue). Scale bar, 100 µm. Quantification of Iba1, GFAP, and NeuN positive cells in Trpc1 KD and Control mice brain was shown in the right panel (n = 4 mice per group). E Quantification of regional cerebral blood flow (rCBF) of Control and Trpc1 KD mice throughout MCAO/R procedure (n = 4 mice per group). * * P < 0.01. n.s.: no significant; KD: Trpc1 KD; CTR: Control.

Article Snippet: Microglia Trpc1 conditional knockdown (Trpc1 KD) mice were generated by mating Trpc1flox/flox (Cyagen Incorporated, Suzhou, China) and CX3C chemokine receptor 1 (Cx3cr1)CreER (Cyagen Incorporated) mice of C57BL/6 background.

Techniques: Knockdown, Isolation, FACS, Western Blot, Control, Expressing, Comparison, Immunofluorescence, Staining, Marker, Binding Assay

Journal: Brain research bulletin

Article Title: Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke.

doi: 10.1016/j.brainresbull.2024.110894

Figure Lengend Snippet: Fig. 3. Microglia-specific Canonical transient receptor potential channel 1 (Trpc1) knockdown reduces brain injury after middle cerebral artery occlusion reperfusion (MCAO/R). A Modified neurological severity score (mNSS) of indicated groups (n = 8 mice per group). B Foot fault rate of the left forelimb of indicated groups (n = 8 mice per group). C Latency to falls in the rotated cylinder (40 revolutions/min) of indicated groups. D Infarct volumes were determined by triphenyl tetrazolium chloride staining in indicated groups. The red area indicates the non-ischemic tissue, white area indicates the infarct area. Infarct volume percentages were quantified by using Photoshop CC 2019 software in the right panel (n = 6 mice per group). E Impaired blood brain barrier integrity was detected by Evans blue staining in indicated groups. The left panel shows the representative staining of brain slices, blue area indicates the dye leakage tissue. The right panel indicates the quantitative determination of Evans blue content in the hemispheric. Details method will be found in the material and methods section (n = 6 mice per group). * * P < 0.01. KD: microglia-specific Trpc1 knockdown; CTR: Control.

Article Snippet: Microglia Trpc1 conditional knockdown (Trpc1 KD) mice were generated by mating Trpc1flox/flox (Cyagen Incorporated, Suzhou, China) and CX3C chemokine receptor 1 (Cx3cr1)CreER (Cyagen Incorporated) mice of C57BL/6 background.

Techniques: Knockdown, Modification, Staining, Software, Control

Journal: Brain research bulletin

Article Title: Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke.

doi: 10.1016/j.brainresbull.2024.110894

Figure Lengend Snippet: Fig. 4. Microglia-specific Canonical transient receptor potential channel 1 (Trpc1) knockdown reduces apoptotic cells caused by middle cerebral artery occlusion reperfusion (MCAO/R). TdT-mediated dUTP nick end labeling (TUNEL, red) staining was used to observe the apoptosis cells in the peri-infarct area of Control and microglia-specific Trpc1 knockdown (Trpc1 KD) mice after MCAO/R. 4’,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei (blue), Scale bars, 100 µm. TUNEL-positive cells numbers quantified by using ImageJ software were shown in the right panel (n = 6 mice per group). ** P < 0.01. KD: Trpc1 KD; CTR: Control.

Article Snippet: Microglia Trpc1 conditional knockdown (Trpc1 KD) mice were generated by mating Trpc1flox/flox (Cyagen Incorporated, Suzhou, China) and CX3C chemokine receptor 1 (Cx3cr1)CreER (Cyagen Incorporated) mice of C57BL/6 background.

Techniques: Knockdown, End Labeling, TUNEL Assay, Staining, Control, Software

Journal: Brain research bulletin

Article Title: Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke.

doi: 10.1016/j.brainresbull.2024.110894

Figure Lengend Snippet: Fig. 5. Neutrophil infiltration after stroke is attenuated with Canonical transient receptor potential channel 1 (Trpc1) knockdown in microglia. A Counts of leukocyte subtypes assayed by flow cytometry in peri-infarct area of microglia-specific Trpc1 knockdown (Trpc1 KD) and Control mice 24 h after middle cerebral artery oc clusion reperfusion (MCAO/R) (n = 4 mice per group). B Counts of leukocyte subtypes assayed by flow cytometry in the spleen of Trpc1 KD and Control mice 24 h after MCAO/R (n = 4 mice per group). C Representative images of immunofluorescence staining of neutrophils marker, lymphocyte antigen 6 complex, locus G (Ly6G, red) from peri-infarct area of Trpc1 KD and Control mice after MCAO/R. Nuclei were stained with 4’,6-diamidino-2-phenylindole (blue). Scale bar, 20 µm. Quantification of neutrophils in indicated groups was shown in the right panel (n = 4 mice per group). D Counts of neutrophils assayed by flow cytometry in peri- infarct area of Trpc1 KD and Control mice 1, 3, 5, and 7 days after MCAO/R (n = 4 mice per group). E Counts of neutrophils assayed by flow cytometry in the spleen of Trpc1 KD and Control mice 1, 3, 5, and 7 days after MCAO/R (n = 4 mice per group). * P < 0.05, ** P < 0.01. n.s.: no significant; KD: Trpc1 KD; CTR: Control.

Article Snippet: Microglia Trpc1 conditional knockdown (Trpc1 KD) mice were generated by mating Trpc1flox/flox (Cyagen Incorporated, Suzhou, China) and CX3C chemokine receptor 1 (Cx3cr1)CreER (Cyagen Incorporated) mice of C57BL/6 background.

Techniques: Knockdown, Flow Cytometry, Control, Immunofluorescence, Staining, Marker

Journal: Brain research bulletin

Article Title: Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke.

doi: 10.1016/j.brainresbull.2024.110894

Figure Lengend Snippet: Fig. 6. Canonical transient receptor potential channel 1 (Trpc1) knockdown in microglia ameliorating stroke outcome via decreasing neutrophil infiltration. Neutrophil in peripheral blood was depleted by intraperitoneal injection of anti- lymphocyte antigen 6 complex, locus G monoclonal antibody in microglia-specific Trpc1 knockdown (Trpc1 KD) and Control mice, middle cerebral artery occlusion reperfusion (MCAO/R) was performed 24 h later. A Modified neurological severity score (mNSS) of indicated groups (n = 8 mice per group). B Foot fault rate of the left forelimb of indicated groups (n = 8 mice per group). C Latency to falls in the rotated cylinder (40 revolutions/min) of indicated groups (n = 8 mice per group). D Infarct volumes were determined by triphenyl tetrazolium chloride staining in indicated groups. The red area indicates the non-ischemic tissue, white area indicates the infarct area. Infarct volume percentages were quantified by using Photoshop CC 2019 software in the right panel (n = 6 mice per group). n.s.: no significant; KD: Trpc1 KD; CTR: Control.

Article Snippet: Microglia Trpc1 conditional knockdown (Trpc1 KD) mice were generated by mating Trpc1flox/flox (Cyagen Incorporated, Suzhou, China) and CX3C chemokine receptor 1 (Cx3cr1)CreER (Cyagen Incorporated) mice of C57BL/6 background.

Techniques: Knockdown, Injection, Control, Modification, Staining, Software

Journal: Brain research bulletin

Article Title: Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke.

doi: 10.1016/j.brainresbull.2024.110894

Figure Lengend Snippet: Fig. 7. Canonical transient receptor potential channel 1 (Trpc1) knockdown in microglia reduces neutrophils infiltration via decreasing CC-Chemokines ligand (CCL) 5and CCL2. A Heat map shows the cytokine/chemokine expression profiles of magnetic-activated cell sorted (MACS) microglia from microglia-specific Trpc1 knockdown (Trpc1 KD) and Control mice 24 h after middle cerebral artery occlusion reperfusion (MCAO/R) (n = 6 mice per group). B CCL5 and CCL2 expression levels were detected by enzyme linked immunosorbent assay (ELISA) in MCAS microglia from Trpc1 KD and Control mice 24 h after MCAO/R. C CCL5 and CCL2 expression levels were detected by ELISA in primary cultured microglia from Trpc1 KD and Control mice upon oxygen-glucose deprivation/regeneration (OGD/R) condition (n = 6 per group). D, E Flow cytometry analysis of CCL5-Cy3 (D) and CCL2-Cy3 (E) signal in MACS microglia from Trpc1 KD Control mice 24 h after MCAO/R. Gates were set by using Fluorescence Minus One (FMO) controls. Gray area: FMO control; White area: positive sample. Quantification of flow cytometry was shown in the right panel (n = 4 per group). Two thousand cells were assayed for each experiment. F Illustration of the co-culture and transmigration system in vitro. The former contains primary microglia from Trpc1 KD and Control mice in the lower chamber and neutrophils in the upper chamber. Co-culture media in the microglia chamber are used as conditioned media to stimulate neutrophils migration. G Quantification of neutrophils migrating towards the lower chamber assayed by flow cytometry (n = 4 per group). H Antibodies against CCL5 or/and CCL2 were added to the microglia medium, and the migration rate of neutrophils was measured in the Transwell system by flow cytometry (n = 4 per group). ** P < 0.01, n.s.: no significant. KD: Trpc1 KD; CTR: Control.

Article Snippet: Microglia Trpc1 conditional knockdown (Trpc1 KD) mice were generated by mating Trpc1flox/flox (Cyagen Incorporated, Suzhou, China) and CX3C chemokine receptor 1 (Cx3cr1)CreER (Cyagen Incorporated) mice of C57BL/6 background.

Techniques: Knockdown, Expressing, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Fluorescence, Co-Culture Assay, Transmigration Assay, In Vitro, Migration

Journal: Brain research bulletin

Article Title: Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke.

doi: 10.1016/j.brainresbull.2024.110894

Figure Lengend Snippet: Fig. 9. Scheme of proposed mechanisms explaining microglia Canonical transient receptor potential channel 1 (TRPC1) aggravates cerebral ischemia and reperfusion injury by inducing neutrophil infiltration through Ca2+/calcineurin/ nuclear factor of activated T-cells c1 (NFATc1) signaling pathway, which stimulates the CC- Chemokines ligand (CCL) 5 and CCL2 expression.

Article Snippet: Microglia Trpc1 conditional knockdown (Trpc1 KD) mice were generated by mating Trpc1flox/flox (Cyagen Incorporated, Suzhou, China) and CX3C chemokine receptor 1 (Cx3cr1)CreER (Cyagen Incorporated) mice of C57BL/6 background.

Techniques: Expressing

Journal: Cell Death Discovery

Article Title: Bcl-2 regulates store-operated Ca 2+ entry to modulate ER stress-induced apoptosis

doi: 10.1038/s41420-018-0039-4

Figure Lengend Snippet: a Control vector (C)-, wild-type Bcl-2 (WT)-, and Bcl-2 mutant (mt)-overexpressing MDCK cells were treated with DMSO or TG for 5 min. Subsequently, immunofluorescence staining was obtained to label STIM1 and nucleus. Representative fluorescence images were obtained using confocal microscopy (scale bar, 20 μm). Arrows indicate the translocation of STIM1 to the juxta-plasma membrane region. b , c Pre-incubation of cells with 2 μM fura-2/AM at 37 °C for 30 min for cytosolic Ca 2+ measurement using a single cell fluorimeter. Depletion of ER lumen-resident Ca 2+ was induced by treating cells in Ca 2+ -free buffer with 2 μM TG for 10 min. The subsequent elevation of Ca 2+ indicated that SOCE occurred during the exchange of Ca 2+ -free buffer to 2 mM-Ca 2+ buffer. b The data in representative curves for the measurement of SOCE are represented as mean ± SEM (where, n ≥ 60 cells) from three independent experiments. c Quantitative analysis of the changes in the peak Ca 2+ levels. All values are represented as mean ± SEM, and the data were found to be statistically significant at p < 0.001 (indicated by ***) compared to control vector (C)-overexpressing cells (Student’s t -test). d Western blotting of SOCE related molecules, such as ER Ca 2+ sensors (STIM1 and STIM2), plasma membrane Ca 2+ channels (Orai1, Orai2, Orai3, and TRPC1), and the internal control β-actin. e Cells were pre-incubated with 2 mM EGTA for 30 min and treated with DMSO or 2 μM TG for 24 h. Representative images of cells were obtained under a bright-field microscope (scale bar, 100 μm). f Bcl-2 mutant (mt)-overexpressing cells were pre-incubated with 20 μM BAPTA/AM, 2 mM EGTA, or 2 μM 2-APB for 30 min and treated with DMSO or 2 μM TG for 24 h. Quantitative analysis of the apoptosis ratio was assessed from the hypodiploid DNA peak of propidium iodide (PI)-stained cells from three independent experiments by flow cytometry. The data were found to be statistically significant at p < 0.01 (indicated by **) compared to the DMSO control (Student’s t -test)

Article Snippet: Cell lysates were harvested in RIPA buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris at pH 7.4, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and Complete TM ), and the lysates were analyzed by Western blotting using antibodies against Bcl-2 (DAKO, Grostrup, Denmark), Bax, Bak, calnexin, SERCA2, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), porin (Molecular Probes, Eugene, OR), caspase-8, caspase-9, caspase-12, SERCA3, pSer70-Bcl-2, pThr167-Bax (Cell Signaling Technology, Beverly, MA), Grp78, STIM1, STIM2, IP3R3 (BD, Franklin Lakes, NJ), Orai1, Orai2, Orai3, and TRPC1 (ProSci, Poway, CA).

Techniques: Control, Plasmid Preparation, Mutagenesis, Immunofluorescence, Staining, Fluorescence, Confocal Microscopy, Translocation Assay, Clinical Proteomics, Membrane, Incubation, Western Blot, Microscopy, Flow Cytometry