Journal: Circulation Research

Article Title: Transient Receptor Potential Channels Contribute to Pathological Structural and Functional Remodeling After Myocardial Infarction

doi: 10.1161/circresaha.115.303831

Figure Lengend Snippet: Figure 5. Transient receptor potential canonical (TRPC) channels colocalize with l-type Ca2+ channel (LTCC) in caveolae membrane microdomains where their organization is required for hypertrophic signaling. A, Plasma membranes (PMs) were purified from total cell homogenates (H) of isolated myocytes from dominant-negative TRPC4 mice. Immunoprecipitations (IPs) and Westerns were performed with the indicated antibodies (B indicates bound fraction; and U, unbound fraction). B, Sucrose density gradient fractionation on purified PMs from isolated adult feline myocytes (AFMs) infected with Ad-TRPC3 confirms the presence of LTCC and TRPC3 channels in caveolin-3 (Cav3)–enriched lipid raft membrane fractions along with the hypertrophic effector calcineurin (Cn; fraction 1 to fraction 11, F1–F11). C, AFMs were infected with Ad-NFAT-GFP and the indicated adenoviruses and NFAT translocation was monitored in response to the TRPC agonist OAG (10 umol/L) in the presence or absence of methyl-β-cyclodextrin (MβCD; 10 mmol/L). Scale bar is 10 μm. Average data are represented in D as the nuclear to cytoplasmic GFP ratio of n=100 cells per condition. P<0.05 was considered significant with *P≤0.05; **P≤0.001; ***P≤0.001 vs red fluorescent protein control; ##P≤0.001; ###P≤0.001 vs TRPC3; &&P≤0.001; &&&P≤0.001 vs TRPC6.

Article Snippet: Sheep anti-rabbit IgG-coated magnetic beads (Dynal Biotech) were pre-incubated with an antibody for TRPC4 (Alomone Labs), TRPC6 (Alomone Labs) or Cav1.2 (LTCC α1C, Millipore) for 2-4 hours at room temperature.

Techniques: Membrane, Clinical Proteomics, Purification, Isolation, Dominant Negative Mutation, Fractionation, Infection, Translocation Assay, Control

Journal: Pulmonary Circulation

Article Title: Stim-activated TRPC-ORAI channels in pulmonary hypertension induced by chronic intermittent hypoxia

doi: 10.1177/2045894020941484

Figure Lengend Snippet: Primers sequence’s characteristics uses in the analysis.

Article Snippet: After appropriate blocking (5% nonfat dried milk) the blots were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076), TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz, sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry milk overnight at 4°C.

Techniques: Sequencing

Journal: Pulmonary Circulation

Article Title: Stim-activated TRPC-ORAI channels in pulmonary hypertension induced by chronic intermittent hypoxia

doi: 10.1177/2045894020941484

Figure Lengend Snippet: Effect of CIH on pulmonary relative gene expression of STOC-forming subunits in rats. Pulmonary gene expression of (a) TRPC1, (b) TRPC4, (c) TRPC6, (d) ORAI1, and (e) ORAI 2 of control (empty bar, n = 6), 14d-CIH (lined bar, n = 6), 21d-CIH (gray bar, n = 7), and 28d-CIH (black bar, n = 7) animals. Values are expressed as 2−ΔΔCT in relation to the control. Values are mean ± SE. * p < 0.05 vs. control, † p < 0.05 vs. all. One-way ANOVA and Newman–Keuls post-test.

Article Snippet: After appropriate blocking (5% nonfat dried milk) the blots were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076), TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz, sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry milk overnight at 4°C.

Techniques: Gene Expression, Control

Journal: Pulmonary Circulation

Article Title: Stim-activated TRPC-ORAI channels in pulmonary hypertension induced by chronic intermittent hypoxia

doi: 10.1177/2045894020941484

Figure Lengend Snippet: Effect of the exposure to 28 days to CIH of the protein levels on STOC-forming subunits in the lungs. Quantification of the protein levels of (a) TRPC1, (b) TRPC6, (c) STIM1, (d) ORAI2, and (e) ORAI1 on control (empty bar, n = 6) and CIH-28d (black bar, n = 6) animals. Values are expressed as a ratio of β-actin. Values are mean ± SE. * p < 0.05 vs. control. Student’s unpaired t test.

Article Snippet: After appropriate blocking (5% nonfat dried milk) the blots were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076), TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz, sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry milk overnight at 4°C.

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: The store-operated Ca 2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCβ2

doi: 10.1016/j.jbc.2023.102882

Figure Lengend Snippet: TRPC1 and Orai3 are involved in carbachol (CCh)-evoked NF-κB activation in Orai1-KO cells. A, Orai1-KO HEK-293 cells (O1KO) transfected with either shTRPC1 (O1KO (shT1)), shTRPC6 (O1KO (shT6)), shOrai3 (O1KO (shO3)), or scramble plasmid (O1KO) were transfected with pNL3.2.NFkB-RE[NlucP/NF-kB-RE/Hygro]. Forty-eight hours later, cells were suspended in HBS containing 1 mM Ca 2+ and then stimulated for 5 h with 100 μM CCh or the vehicle (control) and lysed. Luciferase activity of the lysates was measured as described in the section. The luminescence in relative light units (RLUs) of unstimulated O1KO, O1KO(shT1), O1KO(shT6), and O1KO(shO3) HEK-293 cells were 598,259 ± 47,521, 601,025 ± 51,248, 599,987 ± 32,581, and 601,277 ± 39,985, respectively). B, O1KO, O1KO (shT1), and O1KO (shT6) HEK-293 cells were lysed and then subjected to 10% SDS-PAGE and Western blotting with anti-TRPC1 ( left panels ) or anti-TRPC6 ( right panels ) antibody. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of TRPC1 and TRPC6 bands was normalized to β-actin and quantified ( bar graph ). C, WT HEK-293 cells (WT), WT cells transfected with shOrai3 (shO3), O1KO cells, O1KO cells transfected with shOrai3 (O1KO (shO3)), and Orai1/Orai2/Orai3 triple-KO cells (TKO) transfected with pNL3.2.NFkB-RE[NlucP/NF-kB-RE/Hygro]. Forty-eight hours later, cells were suspended in HBS containing 1 mM Ca 2+ and stimulated for 5 h with 100 μM CCh or the vehicle (control) and lysed. Luciferase activity was measured as described in the section. The luminescence in RLUs of unstimulated WT, O1KO, O1KO(shO3), and TKO HEK-293 cells were 610,982 ± 48,875, 607,588 ± 47,510, 592,115 ± 37,514, and 597,323 ± 37,899, respectively. D, WT HEK-293 cells (WT) and WT cells transfected with shOrai3 (shO3) were lysed and subjected to 10% SDS-PAGE and Western blotting with anti-Orai3 antibody. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of Orai3 bands was normalized to β-actin and quantified ( bar graph ). E, WT, O1KO, TKO, and O1KO (shO3) HEK-293 cells were lysed and then subjected to 10% SDS-PAGE and Western blotting with anti-Orai3. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of Orai3 bands was normalized to β-actin and quantified ( bar graph ). For A, from left to right , n = 18, 14, 18, 13, 18, 12, 18, and 13; for C, from left to right , n = 30, 24, 12, 12, 18, 13, 18, 14, 18, and 12; n values correspond to separate experiments. Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn's test). ∗ p < 0.05 and ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as compared with CCh-treated O1KO HEK-293 cells ( A ) or CCh-treated WT HEK-293 cells ( C ). $ p < 0.05, $$ p < 0.01, and $$$$ p < 0 .0001 as compared with their respective control (untreated) cells. HBS, Hepes-buffered saline; HEK-293, human embryonic kidney 293 cell line.

Article Snippet: Rabbit polyclonal anti-TRPC6 antibody (catalog number: TA328771, epitope: amino acid residues 573–586 of TRPC6) was from Origene Technologies, Inc. Rabbit monoclonal anti-PKCβ2 antibody (clone Y125, epitope located in the region near the C terminus of PKCβ2; catalog number: ab32026) and rabbit monoclonal anti-Orai3 antibody (clone EPR22575-17, catalog number: ab254260) were purchased from Abcam.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, SDS Page, Western Blot, Saline

Journal: ACS Nano

Article Title: A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing

doi: 10.1021/acsnano.3c09814

Figure Lengend Snippet: TRPC6 is impaired in the diabetic wound. (A, B) Fura-2 AM (5 μ M) detected cytosolic Ca 2+ levels in DFs when treated with AGE-BSA. (C) Schematic diagram of calcium channels expressed in the skin. (D) Quantitative real-time PCR from three independent experiments showing the expression levels of TRPC6 in DFs treated with AGE-BSA for 48 h. (E) Western blotting analysis from three independent tests showed the levels of TRPC6 in DFs treated with AGE-BSA for 48 h. (F) Dox diagram of TRPC6 expression. (G) Heat map of correlation between TRPC6 and other genes (Bubble size represents correlation r value; “*” indicates a significant correlation; red indicates positive correlation). (H) Immunohistochemical staining for TRPC6 in skin tissues from surgical specimens of diabetic ulcer patients. Scale bar: 100 μ m. Data are presented as mean ± standard error of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The primary antibodies are as follows: β -actin (Abclonal, #AC026, 1:8000), TRPC6 (Novus, #NBP1-77260, 1:500), α -SMA (HUABIO, #ET1607-53, 1:500), Collagen I (Bioss, #bs-10423R, 1:500), Vimentin (Bioworld, #BS1491, 1:500), SLC8B1 (HUABIO, #ER1901-93, 1:500), MICU1 (HUABIO, #ER1803-99, 1:500), MCU (HUABIO, #ER1803-57, 1:500), SP2 (abcam, #ab229468, 1:500), CD63 (abcam, #ab271286, 1:500), TSG101 (abcam, #ab125011, 1:500), CD81 (abcam, #ab79559, 1:500), CD9 (abcam, #ab236630, 1:500), ALIX (abcam, #ab117600, 1:500), HSP70 (abcam, #ab2787, 1:500), Calnexin (Abclonal, #A15631, 1:500), PCNA (CST, #13110, 1:500), and Histone H3 (CST, #4620).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: ACS Nano

Article Title: A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing

doi: 10.1021/acsnano.3c09814

Figure Lengend Snippet: TRPC6 knockdown decreases Ca 2+ influx and impairs the biological function of DFs in wound healing. (A–C) Fura-2 AM (5 μ M) and Fluo-4 AM (5 μ M) detected cytosolic Ca 2+ levels in DFs when treated with small interfering RNA of TRPC6 (Si-TRPC6) for 48 h. (D) Immunofluorescence analysis showed the level of α -SMA and collagen I of DFs when transfected with Si-TRPC6 for 48 h. Scale bar: 100 μ m. (E) Western blotting analysis of α -SMA, collagen I expression in DFs treated with Si-TRPC6 for 48 h. (F) Quantitative real-time PCR analysis of the expression levels of IL-1β, IL-6, and TNF-α in DFs when treated with Si-TRPC6 for 48 h. (G) Quantitative real-time PCR analysis of the expression levels of TRPC6 in skin tissues when infected in shTRPC6 for 4 weeks. (H) Western blotting analysis of TRPC6 in skin tissues when infected in shTRPC6 for 4 weeks. (I, J) HE and Masson staining evaluated wound and collagen distribution in skin tissues when infected in shTRPC6 for 4 weeks. Scale bar: 100 μ m. (K) Immunohistochemical staining evaluated the expression of CD31. Scale bar: 100 μ m. Data are presented as mean ± standard error of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The primary antibodies are as follows: β -actin (Abclonal, #AC026, 1:8000), TRPC6 (Novus, #NBP1-77260, 1:500), α -SMA (HUABIO, #ET1607-53, 1:500), Collagen I (Bioss, #bs-10423R, 1:500), Vimentin (Bioworld, #BS1491, 1:500), SLC8B1 (HUABIO, #ER1901-93, 1:500), MICU1 (HUABIO, #ER1803-99, 1:500), MCU (HUABIO, #ER1803-57, 1:500), SP2 (abcam, #ab229468, 1:500), CD63 (abcam, #ab271286, 1:500), TSG101 (abcam, #ab125011, 1:500), CD81 (abcam, #ab79559, 1:500), CD9 (abcam, #ab236630, 1:500), ALIX (abcam, #ab117600, 1:500), HSP70 (abcam, #ab2787, 1:500), Calnexin (Abclonal, #A15631, 1:500), PCNA (CST, #13110, 1:500), and Histone H3 (CST, #4620).

Techniques: Knockdown, Small Interfering RNA, Immunofluorescence, Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Infection, Staining, Immunohistochemical staining

Journal: ACS Nano

Article Title: A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing

doi: 10.1021/acsnano.3c09814

Figure Lengend Snippet: HucMSC-sEVs restore TRPC6 expression in vitro and in vivo . (A) Quantitative real-time PCR from three independent experiments showing the expression levels of TRPC6 in DFs treated with hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (B) Western blotting analysis of TRPC6 expression in DFs treated with hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (C) Immunofluorescence analysis showed the location and level of TRPC6 (red) in DFs when treated with DIO-labeled hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Scale bar: 10 μ m. (D) Immunohistochemical staining evaluated the expression of TRPC6 in diabetic rats’ skin. Scale bar: 50 μ m. (E) Immunofluorescent staining for the expression of TRPC6 in diabetic rats’ skin. Scale bar: 100 μ m. (F–H) Fura-2 AM (5 μ M) and Fluo-4 AM (5 μ M) detected cytosolic Ca 2+ levels in DFs when treated with hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Data are presented as mean ± standard error of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The primary antibodies are as follows: β -actin (Abclonal, #AC026, 1:8000), TRPC6 (Novus, #NBP1-77260, 1:500), α -SMA (HUABIO, #ET1607-53, 1:500), Collagen I (Bioss, #bs-10423R, 1:500), Vimentin (Bioworld, #BS1491, 1:500), SLC8B1 (HUABIO, #ER1901-93, 1:500), MICU1 (HUABIO, #ER1803-99, 1:500), MCU (HUABIO, #ER1803-57, 1:500), SP2 (abcam, #ab229468, 1:500), CD63 (abcam, #ab271286, 1:500), TSG101 (abcam, #ab125011, 1:500), CD81 (abcam, #ab79559, 1:500), CD9 (abcam, #ab236630, 1:500), ALIX (abcam, #ab117600, 1:500), HSP70 (abcam, #ab2787, 1:500), Calnexin (Abclonal, #A15631, 1:500), PCNA (CST, #13110, 1:500), and Histone H3 (CST, #4620).

Techniques: Expressing, In Vitro, In Vivo, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Labeling, Immunohistochemical staining, Staining

Journal: ACS Nano

Article Title: A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing

doi: 10.1021/acsnano.3c09814

Figure Lengend Snippet: HucMSC-sEVs transfer transcription factor SP2 to DFs and activate TRPC6 gene expression. (A) Diagram of a database cross to confirm transcription factor SP2. (B) Western blotting analysis of SP2 expression in hucMSC-sEVs. (C) Western blotting analysis of SP2 expression in the nucleus and cytoplasm of DFs when treated with hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (D) Double luciferase reporter gene assay showed SP2 binding to the TRPC6 promoter. (E) ChIP analysis of SP2 binding to the TRPC6 promoter. (F) Schematic structure of the full-length TRPC6 promoter-reporter and its deletion mutant constructs. (G, H) Double luciferase reporter gene assay showed the SP2 binding sites. (I) Quantitative real-time PCR analysis of the expression levels of TRPC6 in DFs when treated with SiSP2-hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (J) Western blotting analysis of TRPC6 and collagen I expression in DFs when treated with SiSP2-hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (K) CCK-8 assay showed proliferation of DFs treated with SiSP2-hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (L) Immunofluorescence analysis showed the level of collagen I of DFs when treated with SiSP2-hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Scale bar: 100 μ m. Data are presented as mean ± standard error of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The primary antibodies are as follows: β -actin (Abclonal, #AC026, 1:8000), TRPC6 (Novus, #NBP1-77260, 1:500), α -SMA (HUABIO, #ET1607-53, 1:500), Collagen I (Bioss, #bs-10423R, 1:500), Vimentin (Bioworld, #BS1491, 1:500), SLC8B1 (HUABIO, #ER1901-93, 1:500), MICU1 (HUABIO, #ER1803-99, 1:500), MCU (HUABIO, #ER1803-57, 1:500), SP2 (abcam, #ab229468, 1:500), CD63 (abcam, #ab271286, 1:500), TSG101 (abcam, #ab125011, 1:500), CD81 (abcam, #ab79559, 1:500), CD9 (abcam, #ab236630, 1:500), ALIX (abcam, #ab117600, 1:500), HSP70 (abcam, #ab2787, 1:500), Calnexin (Abclonal, #A15631, 1:500), PCNA (CST, #13110, 1:500), and Histone H3 (CST, #4620).

Techniques: Gene Expression, Western Blot, Expressing, Luciferase, Reporter Gene Assay, Binding Assay, Mutagenesis, Construct, Real-time Polymerase Chain Reaction, CCK-8 Assay, Immunofluorescence

Journal: ACS Nano

Article Title: A Dual Role of Mesenchymal Stem Cell Derived Small Extracellular Vesicles on TRPC6 Protein and Mitochondria to Promote Diabetic Wound Healing

doi: 10.1021/acsnano.3c09814

Figure Lengend Snippet: HucMSC-sEVs protect the function of mitochondria to maintain calcium balance. (A) Quantitative real-time PCR analysis of the expression levels of TRPC6 in DFs transfected with overexpression plasmid of TRPC6 for 48 and 96 h. (B) Calcium ion assay of DFs treated with AGE-BSA, overexpression plasmid of TRPC6 (OE-TRPC6), overexpression of controlled plasmid (OE-Vector), CaCl 2 (2 mM), and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 24, 48, 72, and 96 h. (C) Annexin V/PI staining of DFs treated with AGE-BSA, overexpression plasmid of TRPC6 (OE-TRPC6), overexpression of controlled plasmid (OE-Vector), CaCl 2 (2 mM), and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 24, 48, 72, and 96 h. (D) The wound healing assay showed migration of DFs when treated with AGE-BSA, overexpression plasmid of TRPC6 (OETRPC6), overexpression of controlled plasmid (OE-Vector), CaCl 2 (2 mM), and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 24, 48, 72, and 96 h. (E) Mitochondrial membrane potential assay with JC-1 of DFs treated with AGE-BSA and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Scale bar: 100 μ m. (F) Western blotting analysis of MCU, MICU1, and NCLX expression in DFs treated with AGE-BSA and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. (G) Fluorescent Ca 2+ indicator of Rhod-2 AM (5 μ M) showed mitochondrial calcium level in DFs treated with AGE-BSA and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Scale bar: 200 μ m. (H) Relative mitochondrial Ca 2+ uptake in DFs treated with AGE-BSA and hucMSC-sEVs (1.0 × 10 10 particles/mL) for 48 h. Data are presented as mean ± standard error of the mean (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The primary antibodies are as follows: β -actin (Abclonal, #AC026, 1:8000), TRPC6 (Novus, #NBP1-77260, 1:500), α -SMA (HUABIO, #ET1607-53, 1:500), Collagen I (Bioss, #bs-10423R, 1:500), Vimentin (Bioworld, #BS1491, 1:500), SLC8B1 (HUABIO, #ER1901-93, 1:500), MICU1 (HUABIO, #ER1803-99, 1:500), MCU (HUABIO, #ER1803-57, 1:500), SP2 (abcam, #ab229468, 1:500), CD63 (abcam, #ab271286, 1:500), TSG101 (abcam, #ab125011, 1:500), CD81 (abcam, #ab79559, 1:500), CD9 (abcam, #ab236630, 1:500), ALIX (abcam, #ab117600, 1:500), HSP70 (abcam, #ab2787, 1:500), Calnexin (Abclonal, #A15631, 1:500), PCNA (CST, #13110, 1:500), and Histone H3 (CST, #4620).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Over Expression, Plasmid Preparation, Staining, Wound Healing Assay, Migration, Membrane, Western Blot

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 1. Expression of TRPC6 protein in human glioma tissue. A) Representative immunoblots of total lysates extracted from human glioma (six samples shown) and normal brain (six samples shown) tissues probed with anti–transient re ceptor potential canonical (TRPC)6 (106 kDa) or anti-TRPC3 (97 kDa) antibodies. GAPDH served as the protein loading control. B and C) Quantification of TRPC6 and TRPC3 protein levels in glioma (n = 33) and normal brain (n = 17) tissues from the immunoblots. Each dot repre sents the band density of TRPC6 and 3 proteins normalized to that of the corre sponding GAPDH in each sample. n = number of samples. Means and 95% confidence intervals are represented by open lines and error bars. **P < .001 vs normal, calculated using the two-sided Student t test. D) Representative immu nohistological staining samples of human glioma or normal brain tissues with anti- TRPC6 antibody. a–i) Three glioblastoma multiforme (GBM, grade 4) samples; j–l) anaplastic oligodendrocytoma (grade 3 oligodendrocytoma); m–o) grade 2 astro cytoma; and p–r) normal (para-tumor) tissues. Sections were stained by the 3,3′-diaminobenzidine (DAB) staining method to detect TRPC6 protein in the tissues and were counterstained with hematoxylin to stain the nucleus. Serial sections of the same samples were used for hematoxylin and eosin (HE) staining. Magnified ×40 panels represented the white rectangles in the ×10 panels. Scale bar = 100 µm. E) Bar graph representa tion of TRPC6 immunohistological scores in glioma of different grades. The per centage of sections with different scores (strong, moderate, and weak) in each grade is shown. Grade 4 glioma (GBM), n = 18; grade 3, n = 7; grades 1 and 2 combined, n = 8. n = number of samples. F) Expression of TRPC6 mRNA in glioma and normal tissues detected by in situ hybridization. Immunostaining by anti- TRPC6 antibody (DAB, ×40) on serial sections of the same samples used for immunohistological staining. Sense probe (×40) was used as a negative con trol, and the antisense probe (×40) was used to detect the TRPC6 mRNA. Scale bar = 100 µm.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Expressing, Western Blot, Control, Staining, In Situ Hybridization, Immunostaining

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 2. Functional activity of transient receptor potential canonical 6 (TRPC6) channels in glioma cells. Fura-2 AM imaging showing induc tion and attenuation of transient elevation of [Ca2+]i in U87 cells. R refers to the ratio of the emission at 500 nm induced by 340 and 380 nm exci tation, captured at 6-second intervals. DR/R was calculated as (R 2 Rbaseline)/Rbaseline, Rbaseline being the mean value of R during pretreatment. The recording time in minutes is shown. Squares represent the mean values. Error bars = 95% confidence intervals. Corresponding bar charts show the area of DR/R curve in the Fura-2 AM imaging, 3 minutes after addition of platelet-derived growth factor-BB (PDGF-BB) or 10 minutes after addition of cyclopiazonic acid (CPA). In all bar charts, the y-axis shows the absolute value of the area divided by 100. Means and upper 95% confidence intervals are shown. All P values were calculated using the two-sided Student t test. Ctrl = control; n = number of cells. A) Induction with 50 ng/mL PDGF-BB (Ctrl, solid square) and attenuation with 20 µM SKF96365 (SKF, open square). Medium was supplemented with calcium (Ca2+). B) Bar chart corresponds to (A). Ctrl, n = 48; SKF, n = 32. *P = .014 vs Ctrl. Data from three independent experiments are shown in (A) and (B). C) Induction with PDGF-BB in cells infected with adenovirus-expressing wild-type TRPC6 (WTC6, solid square) and at tenuation in cells expressing the dominant-negative form of TRPC6 (DNC6, open square). Duration of infection was 48 hours. Medium was supplemented with Ca2+. D) Bar chart corresponds to (C). Cells infected with adenovirus expressing green fluorescent protein (GFP) served as the control. GFP, n = 48; DNC6, n = 51; WTC6, n = 45. **P = .001, GFP vs DNC6, and **P < .001, WTC6 vs DNC6. Data from at least three indepen dent experiments are shown in (C) and (D). E) Induction with PDGF-BB in cells infected with GFP (solid square) or DNC6 (open square) in Ca2+- free medium (0 Ca2+) and Ca2+-containing medium (2 mM Ca2+). Smaller peaks represent the internal Ca2+ release induced by PDGF-BB, and larger peaks represent the extracellular Ca2+ entry on addition of 2 mM

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Functional Assay, Activity Assay, Imaging, Derivative Assay, Control, Infection, Expressing, Dominant Negative Mutation

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 3. Effect of inhibition of transient re ceptor potential canonical 6 (TRPC6) activity or expression on glioma cell growth. A and B) Number of U251 and U87 cells infected with adenovirus expressing green fluorescent protein (GFP), DNC6, or WTC6. Cell numbers were normalized to those at day 0. Means and upper 95% confidence intervals from three independent experiments performed in dupli cate are shown. *P < .05, **P < .01, GFP or WTC6 vs DNC6. C) Representative images of U251 and U87 colonies infected with adeno virus expressing GFP, DNC6, or WTC6. Colonies stained with crystal violet after 14 days of seed ing are shown. D) Quantification of U251 and U87 colonies (shown in C) by measuring the optical density at 595 nm (OD595). OD595 values of DNC6 and WTC6 groups were normalized to that of GFP group. Means and upper 95% confi dence intervals are shown. Data are representa tive of four independent experiments, performed in duplicate. *P < .05, **P < .01, GFP or WTC6 vs DNC6. E) U251 colonies infected with lentivi rus-based scrambled short hairpin RNA (shRNA) (Ctrl), shTRPC6-1 (C6-1), and shTRPC6-2 (C6-2). Colonies stained with crystal violet after 14 days of seeding were shown. Ctrl = control. F) Differential interference contrast (DIC) and fluo rescent (GFP) images of typical single colonies formed by U251 cells infected with the indi cated lentiviral constructs—scrambled shRNA (Ctrl), shTRPC6-1 (C6-1), and shTRPC6-2 (C6-2). The duration of infection was 72 hours before seeding. The lentivirus was GFP tagged. Ctrl = control. Images were taken 14 days after seed ing. Scale bar = 100 µm. Merge panels show the merged DIC and GFP images. G) Quantification of the U251 colonies (shown in E) by measuring the OD595. Means and upper 95% confidence intervals are shown. Data are representative of four independent experiments performed in duplicate. *P = .032, **P < .001 vs Ctrl. All P values were calculated using the two-sided Student t test.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Expressing, Infection, Virus, Staining, shRNA, Control, Construct

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 4. Effect of inhibition of transient receptor potential canonical 6 (TRPC6) activity or expres sion on G2 phase arrest. A and B) Flow cytometric analysis of U251 DNA content after infection with adenovirus expressing green fluorescent protein (GFP), DNC6, and WTC6 for 72 hours or the indi cated lentivirus-based short hairpin RNA (shRNA) constructs (Ctrl, C6-1, and C6-2), 5–10 days after infection. DNA content is shown as 2n and 4n in the x-axis. 2n = cells in G0/G1 phase, and 4n = cells in the G2/M phase. Data are representative of four independent experiments. Ctrl = control, scrambled shRNA (Ctrl); C6-1 = shTRPC6-1 and C6-2 = shTRPC6-2. C) Quantitative real-time poly merase chain reaction and immunoblot analysis (inset) showing knockdown of CACNA1G in U251 cells by lentivirus-based shRNA constructs against the CACNA1G (CACNA1G RNA interference [RNAi]). Control (Ctrl) RNAi was nonsense shRNA. Data are representative of five independent exper iments. TUBA1A served as the protein loading control. Error bar = upper 95% confidence inter val. **P < .001 vs Ctrl RNAi. D) Flow cytometric analysis of U251 cell cycle after infection with the lentivirus-based shRNA construct (CACNA1G RNAi) (shown in C), 5–10 days after infection. Data shown are a representative of six independent experiments. E) Mitotic index of U251 cells after infection with adenovirus expressing GFP, DNC6, and WTC6 for 72 hours. Mitotic index (%) is the percentage of mitotic cells in total cells. Means and upper 95% confidence interval from three independent experiments are shown. **P = .005, GFP vs DNC6, and *P = .022, WTC6 vs DNC6. F) Effect of TRPC6 knockdown on the level of phos phorylated cyclin-dependent kinase 1 at tyrosine15 (P-CDK1 [Y15]). Immunoblot analysis of total U251 cell lysates using the indicated antibodies. Proteins were extracted approximately 10 days after infection. C6-1 and -2 lanes show approxi mately 40% and 60% decrease in TRPC6 level and increased in P-CDK1 (Y15) level by two- or four fold, respectively. TRPC3 blots show that TRPC6 knockdown did not have an effect on its homolog. TUBA1A served as the protein loading control. Data are representative of three independent ex periments. All P values were calculated using the two-sided Student t test.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Infection, Expressing, shRNA, Construct, Control, Western Blot, Knockdown

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 6. Effect of inhibition of transient re ceptor potential canonical 6 (TRPC6) activity on radiosensitization of glioma cells. A) Representative crystal violet staining of the colonies formed by U251 cells 11 days after irradiation with 0, 2, 4, 6, and 8 Gy. Cells were treated with vehicle (Ctrl) or 10 µM SKF96365 (SKF) 24 hours before irradiation. Ctrl = con trol. B and C) Surviving fraction of U251 cells after irradiation with 0, 2, 4, 6, and 8 Gy. Cells were treated with vehicle (Ctrl) or 10 µM SKF96365 (SKF) for 24 hours (B), or infected with adenovirus-based green fluorescent protein (GFP) or DNC6 at multiplicity of infec tion = 2, for 72 hours (C) before irradiation. Ctrl = control. Data were representative of five independent experiments, performed in dupli cate. Error bars = 95% confidence interval. **P = .006, SKF vs Ctrl at 2 Gy; **P = .005, SKF vs Ctrl at 4 Gy; **P < .001, GFP vs DNC6 at 2 Gy; **P = .002, GFP vs DNC6 at 4 Gy. All P values were calculated using the two-sided Student t test.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Staining, Irradiation, Infection, Control

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 7. Effect of inhibition of transient re ceptor potential canonical 6 (TRPC6) activity on the development of xenografted human glioma. A) The tumor volumes in green fluorescent protein (GFP)-, DNC6-, and WTC6-infected groups (n = 7 per group) were determined every 5 days for 30 days after implantation of U87 cells. Means and upper 95% confidence inter vals were shown. *P < .05, **P < .01, GFP or WTC6 vs DNC6. P values were calculated using the two-sided Student t test. B) Representative photographs of nude mice bearing xenografted tumors on day 30 of implantation. C) Scatter diagram of individual tumor volume of xeno grafted tumors on day 30 of implantation. Each symbol represents a single tumor. Error bars represent the interquartile range. *P = .014, GFP vs DNC6, **P < .001 WTC6 vs DNC6. P values were calculated using the two-sided Student t test. D) Survival curve was plotted by Kaplan– Meier analysis. Mice bearing glioma from DNC6-infected U87 cells survived longer than GFP- or WTC6-infected U87 cells. The number of mice (n) in each group is indicated. Each curve was compared using the two-sided log- rank test. P < .001, DNC6 vs GFP or WTC6. Number of mice at risk at each time point is also shown. E) Hematoxylin and eosin staining of representative coronal sections of the mouse brains 20 days after implantation. The tumors were derived from GFP-, DNC6-, and WTC6- infected U87 cells, respectively.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Infection, Staining, Derivative Assay

Journal: Molecular medicine reports

Article Title: Trpc6 knockout protects against renal fibrosis by restraining the CN‑NFAT2 signaling pathway in T2DM mice.

doi: 10.3892/mmr.2023.13136

Figure Lengend Snippet: Figure 1. Trpc6 knockout significantly protects against kidney injury and renal fibrosis in T2DM mice. (A) H&E staining (magnification, x400; scale bar, 20 µm); (B) The pathological scoring of kidney injury. (C) The bands of KIM‑1 and β‑actin; (D) Relative expression of KIM‑1. Results are expressed as mean ± SD, n=4. **P<0.01 vs. WT group; #P<0.05 and ##P<0.01 vs. Trpc6‑/‑ group; &P<0.05 and &&P<0.01 vs. WT + HFD + STZ group. T2DM, type‑2 diabetes mellitus; H&E, hematoxylin and eosin; KIM‑1, kidney injury molecule‑1; WT, wild type; HFD, high fat diet; STZ, streptozotocin.

Article Snippet: Animals and treatment. in the present study, the Trpc6 knockout (Trpc6 ‐/‐) mice (c57Bl/6J) were obtained by Suzhou cyagen Biosciences (Suzhou) inc.

Techniques: Knock-Out, Staining, Expressing

Journal: Molecular medicine reports

Article Title: Trpc6 knockout protects against renal fibrosis by restraining the CN‑NFAT2 signaling pathway in T2DM mice.

doi: 10.3892/mmr.2023.13136

Figure Lengend Snippet: Figure 2. Trpc6 knockout significantly protects against kidney injury and renal fibrosis in T2DM mice. (A) PAS and Masson staining (magnification, x400; scale bar, 20 µm); (B) The mean density of mesangial cells in the glomerulus; (C) The percentage of mesangial area to glomerular area; (D) The density of the positive area in glomeruli over control. Results are expressed as mean ± SD, n=4. **P<0.01 vs. WT group; #P<0.05 and ##P<0.01 vs. Trpc6‑/‑ group; &P<0.05 vs. WT + HFD + STZ group; NS, not significant. T2DM, type‑2 diabetes mellitus; PAS, periodic acid‑Schiff; WT, wild type; HFD, high fat diet; STZ, streptozotocin.

Article Snippet: Animals and treatment. in the present study, the Trpc6 knockout (Trpc6 ‐/‐) mice (c57Bl/6J) were obtained by Suzhou cyagen Biosciences (Suzhou) inc.

Techniques: Knock-Out, Staining, Control

Journal: Molecular medicine reports

Article Title: Trpc6 knockout protects against renal fibrosis by restraining the CN‑NFAT2 signaling pathway in T2DM mice.

doi: 10.3892/mmr.2023.13136

Figure Lengend Snippet: Figure 4. Trpc6 knockout has no influence on renal ROS generation in T2DM mice. (A) The production of ROS in the renal cortex (DHE staining, magnifi‑ cation, x400; scale bar, 20 µm); (B) Relative ROS production in renal cortex. Results are expressed as mean ± SD, n=4. **P<0.01 vs. WT group; ##P<0.01 vs. Trpc6‑/‑ group; NS, not significant. ROS, reactive oxygen species; T2DM, type‑2 diabetes mellitus; DHE, dihydroethidium; WT, wild type; HFD, high fat diet; STZ, streptozotocin.

Article Snippet: Animals and treatment. in the present study, the Trpc6 knockout (Trpc6 ‐/‐) mice (c57Bl/6J) were obtained by Suzhou cyagen Biosciences (Suzhou) inc.

Techniques: Knock-Out, Staining

Journal: Molecular medicine reports

Article Title: Trpc6 knockout protects against renal fibrosis by restraining the CN‑NFAT2 signaling pathway in T2DM mice.

doi: 10.3892/mmr.2023.13136

Figure Lengend Snippet: Figure 3. Trpc6 knockout clearly improves the glomerular ultrastructure in T2DM mice (transmission electron microscopy; magnification, x10,000; scale bar, 2 µm). T2DM, type‑2 diabetes mellitus; WT, wild type; HFD, high fat diet; STZ, streptozotocin.

Article Snippet: Animals and treatment. in the present study, the Trpc6 knockout (Trpc6 ‐/‐) mice (c57Bl/6J) were obtained by Suzhou cyagen Biosciences (Suzhou) inc.

Techniques: Knock-Out, Transmission Assay, Electron Microscopy

Journal: Molecular medicine reports

Article Title: Trpc6 knockout protects against renal fibrosis by restraining the CN‑NFAT2 signaling pathway in T2DM mice.

doi: 10.3892/mmr.2023.13136

Figure Lengend Snippet: Figure 5. Trpc6 knockout decreases renal fibrosis‑related protein expression in T2DM mice. (A) Immunohistochemical staining (magnification, x400; scale bar, 20 µm); (B) Relative expression of COL4; (C) Relative expression of FN. Results are expressed as mean ± SD, n=4. **P<0.01 vs. WT group; ##P<0.01 vs. Trpc6‑/‑ group; &P<0.05 and &&P<0.01 vs. WT + HFD + STZ group. T2DM, type‑2 diabetes mellitus; COL4, collagen IV; FN, fibronectin; WT, wild type; HFD, high fat diet; STZ, streptozotocin.

Article Snippet: Animals and treatment. in the present study, the Trpc6 knockout (Trpc6 ‐/‐) mice (c57Bl/6J) were obtained by Suzhou cyagen Biosciences (Suzhou) inc.

Techniques: Knock-Out, Expressing, Immunohistochemical staining, Staining

Journal: Molecular medicine reports

Article Title: Trpc6 knockout protects against renal fibrosis by restraining the CN‑NFAT2 signaling pathway in T2DM mice.

doi: 10.3892/mmr.2023.13136

Figure Lengend Snippet: Figure 6. Trpc6 knockout decreases the renal expressions of TGF‑β and p‑SMAD2/3 in T2DM mice. (A) The bands of TGF‑β, SMAD2/3, p‑SMAD2/3 and β‑actin; (B) Relative expression of TGF‑β; (C) Relative expression of SMAD2/3; (D) Relative expression of p‑SMAD2/3/SMAD2/3. Results are expressed as mean ± SD, n=4. **P<0.01 vs. WT group; #P<0.05 vs. Trpc6‑/‑ group; &P<0.05 vs. WT + HFD + STZ group; p‑, phosphorylated; T2DM, type‑2 diabetes mellitus; WT, wild type; HFD, high fat diet; STZ, streptozotocin.

Article Snippet: Animals and treatment. in the present study, the Trpc6 knockout (Trpc6 ‐/‐) mice (c57Bl/6J) were obtained by Suzhou cyagen Biosciences (Suzhou) inc.

Techniques: Knock-Out, Expressing

Journal: Molecular medicine reports

Article Title: Trpc6 knockout protects against renal fibrosis by restraining the CN‑NFAT2 signaling pathway in T2DM mice.

doi: 10.3892/mmr.2023.13136

Figure Lengend Snippet: Figure 7. Trpc6 knockout decreases renal NLRP3 inflammasomes in T2DM mice. (A) The bands of NLRP3, Caspase‑1 p20, ASC and β‑actin; (B) Relative expression of NLRP3; (C) Relative expression of ASC; (D) Relative expression of Caspase‑1 p20. Results are expressed as mean ± SD, n=4. **P<0.01 vs. WT group; ##P<0.01 vs. Trpc6‑/‑ group; &P<0.05 and &&P<0.01 vs. WT + HFD + STZ group. NLRP3, Nod‑like receptor protein 3; T2DM, type‑2 diabetes mellitus; WT, wild type; HFD, high fat diet; STZ, streptozotocin; ASC, apoptosis‑associated speck‑like protein containing a CARD.

Article Snippet: Animals and treatment. in the present study, the Trpc6 knockout (Trpc6 ‐/‐) mice (c57Bl/6J) were obtained by Suzhou cyagen Biosciences (Suzhou) inc.

Techniques: Knock-Out, Expressing

Journal: Molecular medicine reports

Article Title: Trpc6 knockout protects against renal fibrosis by restraining the CN‑NFAT2 signaling pathway in T2DM mice.

doi: 10.3892/mmr.2023.13136

Figure Lengend Snippet: Figure 8. Trpc6 knockout inhibits the renal CN‑NFAT2 signaling in T2DM mice. (A) The bands of NFAT2, CN, TRPC6 and β‑actin; (B) Relative expression of TRPC6; (C) Relative expression of CN; (D) Relative expression of NFAT2. Results are expressed as mean ± SD, n=4. **P<0.01 vs. WT group; #P<0.05 vs. Trpc6‑/‑ group; &P<0.05 vs. WT + HFD + STZ group. CN, calcineurin; NFAT, nuclear factor of activated T cells; T2DM, type‑2 diabetes mellitus; TRPC6, transient receptor potential channel 6; WT, wild type; HFD, high fat diet; STZ, streptozotocin.

Article Snippet: Animals and treatment. in the present study, the Trpc6 knockout (Trpc6 ‐/‐) mice (c57Bl/6J) were obtained by Suzhou cyagen Biosciences (Suzhou) inc.

Techniques: Knock-Out, Expressing

Journal: Molecular medicine reports

Article Title: Trpc6 knockout protects against renal fibrosis by restraining the CN‑NFAT2 signaling pathway in T2DM mice.

doi: 10.3892/mmr.2023.13136

Figure Lengend Snippet: Figure 9. Trpc6 knockout alleviates calcium homeostasis disorder in PA + HG‑induced HMCs. (A) The ratio (F340/F380) corresponds to BAPTA and CaCl2 (2 mM) in HMCs. (B) The ratio (F340/F380) of basal [Ca2+]i. (C) The ΔRatio (F340/F380) after BAPTA. (D) The ΔRatio (F340/F380) after CaCl2. Data are expressed as mean ± SD, n=3. **P<0.01 vs. Control group; #P<0.05 and ##P<0.01 vs. PA + HG group. PA, palmitic acid; HG, high glucose; HMCs, human mesangial cells.

Article Snippet: Animals and treatment. in the present study, the Trpc6 knockout (Trpc6 ‐/‐) mice (c57Bl/6J) were obtained by Suzhou cyagen Biosciences (Suzhou) inc.

Techniques: Knock-Out, Control