Journal: American Journal of Physiology - Cell Physiology

Article Title: Flow shear stress enhances intracellular Ca 2+ signaling in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension

doi: 10.1152/ajpcell.00115.2014

Figure Lengend Snippet: Pharmacological blockade of the mechanosensitive channel TRPM7 with 2-aminoethoxydiphenyl borate (2-APB) significantly attenuates the shear stress-induced increase in [Ca2+]cyt in both normal and IPAH-PASMC. A: representative traces showing changes in [Ca2+]cyt in normal (Normal) and IPAH (IPAH) PASMC before and during application of shear stress (Perfusion) in the absence or presence of the TRPM7 (and TRPC6) channel inhibitor 2-APB (20 μM). B: summarized data (means ± SE) showing the amplitude of shear stress-induced transient (Trans) and plateau (Plateau) increases in [Ca2+]cyt in the absence or presence of 20 μM 2-APB in Normal (left) and IPAH (right) PASMC. *P < 0.05 vs. Perfusion. C: representative traces showing changes in [Ca2+]cyt before and during extracellular application of 10 μM bradykinin (BRK) in Normal and IPAH-PASMC or treated by 20 μM 2-APB before and during BRK stimulation in IPAH-PASMC. D: summarized data (means ± SE) showing the amplitude of BRK-induced rises in [Ca2+]cyt with or without treatment by 20 μM 2-APB. *P < 0.05 vs. Normal PASMC and 2-APB-treated IPAH.

Article Snippet: PASMC were transiently transfected with control siRNA (10 μM, sc-37007; Santa Cruz Biotechnology), or TRPM7 siRNA (10 μM, sc-42662; Santa Cruz Biotechnology) or TRPV4 siRNA (10 μM, sc-64726; Santa Cruz Biotechnology) using Xfect siRNA transfection reagent protocol (Clontech Laboratories).

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Flow shear stress enhances intracellular Ca 2+ signaling in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension

doi: 10.1152/ajpcell.00115.2014

Figure Lengend Snippet: Upregulated protein expression of the mechanosensitive channels TRPV4, TRPM7, and TRPC6 in PASMC from IPAH patients compared with PASMC from normal subjects. A: Western blot analysis of TRPV4 and TRPV4 in two normal (Nor) PASMC samples and four IPAH-PASMC samples (left). β-Actin was used as a control. Summarized data (means ± SE) show expression levels of TRPM7 and TRPV4 in Nor and IPAH-PASMC (right). *P < 0.05 vs. Nor. B: Western blot analysis of TRPC6 from one normal PASMC sample and two IPAH-PASMC samples (left), along with the summarized data (means ± SE, right), showing significant upregulation of TRPC6 in IPAH-PASMC compared with normal PASMC. *P < 0.05 vs. Nor.

Article Snippet: PASMC were transiently transfected with control siRNA (10 μM, sc-37007; Santa Cruz Biotechnology), or TRPM7 siRNA (10 μM, sc-42662; Santa Cruz Biotechnology) or TRPV4 siRNA (10 μM, sc-64726; Santa Cruz Biotechnology) using Xfect siRNA transfection reagent protocol (Clontech Laboratories).

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Flow shear stress enhances intracellular Ca 2+ signaling in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension

doi: 10.1152/ajpcell.00115.2014

Figure Lengend Snippet: Downregulation of TRPM7 or TRPV4 with siRNA significantly attenuates shear stress-mediated increases in [Ca2+]cyt in PASMC from IPAH patients. A: Western blot analysis of TRPM7 and TRPV4 in IPAH-PASMC (Control), IPAH-PASMC treated with scrambled siRNA (Cont-siR), and IPAH-PASMC treated with siRNA for TRPM7 (left) or TRPV4 (right). β-Actin was used as a control. B: representative traces showing changes in [Ca2+]cyt during application of flow shear stress (Perfusion) in normal (top) and IPAH (top) PASMC (Control), as well as normal and IPAH-PASMC treated with scrambled siRNA (Cont-siR) and siRNA for TRPM7 (siR-TRPM7) or TRPV4 (siR-TRPV4). C: summarized data (means ± SE) showing the amplitude of shear stress-induced transient (Trans) and plateau (Plateau) phase increases of [Ca2+]cyt in normal PASMC (top, Control) and IPAH-PASMC (top, Control), as well as normal and IPAH-PASMC treated with Cont-siRNA, siR-TRPM7, or siR-TRPV4. *P < 0.05 vs. Control and Cont-siR. The results shown indicate that TRPM7 and TRPV4 are necessary for shear stress-induced increase in [Ca2+]cyt in normal and IPAH-PASMC.

Article Snippet: PASMC were transiently transfected with control siRNA (10 μM, sc-37007; Santa Cruz Biotechnology), or TRPM7 siRNA (10 μM, sc-42662; Santa Cruz Biotechnology) or TRPV4 siRNA (10 μM, sc-64726; Santa Cruz Biotechnology) using Xfect siRNA transfection reagent protocol (Clontech Laboratories).

Techniques: Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Flow shear stress enhances intracellular Ca 2+ signaling in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension

doi: 10.1152/ajpcell.00115.2014

Figure Lengend Snippet: Pharmacological blockade of the mechanosensitive channel TRPV4 with Ruthenium red (RR) significantly attenuates the shear stress-induced increase in [Ca2+]cyt in both normal and IPAH-PASMC. A: representative traces showing changes in [Ca2+]cyt in normal (Normal, top) and IPAH (IPAH, bottom) PASMC before and during application of shear stress (Perfusion) in the absence or presence of the TRPV4 channel inhibitor RR (1 μM). B: summarized data (means ± SE) showing the amplitude of shear stress-induced transient (Trans) and plateau (Plateau) increases of [Ca2+]cyt in the absence (Cont) or presence (RR) of 1 μM RR in Normal (top) and IPAH (bottom) PASMC. *P < 0.05 vs. Cont.

Article Snippet: PASMC were transiently transfected with control siRNA (10 μM, sc-37007; Santa Cruz Biotechnology), or TRPM7 siRNA (10 μM, sc-42662; Santa Cruz Biotechnology) or TRPV4 siRNA (10 μM, sc-64726; Santa Cruz Biotechnology) using Xfect siRNA transfection reagent protocol (Clontech Laboratories).

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Flow shear stress enhances intracellular Ca 2+ signaling in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension

doi: 10.1152/ajpcell.00115.2014

Figure Lengend Snippet: Activation of TRPV4 channels with 4α-phorbol 12,13-didecanoate (4αPDD) causes a greater increase in [Ca2+]cyt in IPAH-PASMC than in normal PASMC. A and B: averaged records obtained from all cells (Averaged; A) and representative records obtained from selected individual cells (Individual; B) showing changes in [Ca2+]cyt before and during extracellular application of 10 μM 4αPDD (a selective agonist of TRPV4 channels) in normal PASMC (Normal, left), IPAH-PASMC (middle), and IPAH-PASMC pretreated with 1 μM Ruthenium red (RR, a blocker of TRPV4 channels, right). C: summarized data (means ± SE) showing the amplitude of 4αPDD-induced increases in [Ca2+]cyt in normal (Nor) and IPAH-PASMC with (RR) or without (Cont) treatment with 1 μM RR. *P < 0.05 vs. Nor PASMC and Cont-IPAH-PASMC.

Article Snippet: PASMC were transiently transfected with control siRNA (10 μM, sc-37007; Santa Cruz Biotechnology), or TRPM7 siRNA (10 μM, sc-42662; Santa Cruz Biotechnology) or TRPV4 siRNA (10 μM, sc-64726; Santa Cruz Biotechnology) using Xfect siRNA transfection reagent protocol (Clontech Laboratories).

Techniques: Activation Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Flow shear stress enhances intracellular Ca 2+ signaling in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension

doi: 10.1152/ajpcell.00115.2014

Figure Lengend Snippet: Upregulated protein expression of the mechanosensitive channels TRPV4, TRPM7, and TRPC6 in PASMC from IPAH patients compared with PASMC from normal subjects. A: Western blot analysis of TRPV4 and TRPV4 in two normal (Nor) PASMC samples and four IPAH-PASMC samples (left). β-Actin was used as a control. Summarized data (means ± SE) show expression levels of TRPM7 and TRPV4 in Nor and IPAH-PASMC (right). *P < 0.05 vs. Nor. B: Western blot analysis of TRPC6 from one normal PASMC sample and two IPAH-PASMC samples (left), along with the summarized data (means ± SE, right), showing significant upregulation of TRPC6 in IPAH-PASMC compared with normal PASMC. *P < 0.05 vs. Nor.

Article Snippet: PASMC were transiently transfected with control siRNA (10 μM, sc-37007; Santa Cruz Biotechnology), or TRPM7 siRNA (10 μM, sc-42662; Santa Cruz Biotechnology) or TRPV4 siRNA (10 μM, sc-64726; Santa Cruz Biotechnology) using Xfect siRNA transfection reagent protocol (Clontech Laboratories).

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Flow shear stress enhances intracellular Ca 2+ signaling in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension

doi: 10.1152/ajpcell.00115.2014

Figure Lengend Snippet: Downregulation of TRPM7 or TRPV4 with siRNA significantly attenuates shear stress-mediated increases in [Ca2+]cyt in PASMC from IPAH patients. A: Western blot analysis of TRPM7 and TRPV4 in IPAH-PASMC (Control), IPAH-PASMC treated with scrambled siRNA (Cont-siR), and IPAH-PASMC treated with siRNA for TRPM7 (left) or TRPV4 (right). β-Actin was used as a control. B: representative traces showing changes in [Ca2+]cyt during application of flow shear stress (Perfusion) in normal (top) and IPAH (top) PASMC (Control), as well as normal and IPAH-PASMC treated with scrambled siRNA (Cont-siR) and siRNA for TRPM7 (siR-TRPM7) or TRPV4 (siR-TRPV4). C: summarized data (means ± SE) showing the amplitude of shear stress-induced transient (Trans) and plateau (Plateau) phase increases of [Ca2+]cyt in normal PASMC (top, Control) and IPAH-PASMC (top, Control), as well as normal and IPAH-PASMC treated with Cont-siRNA, siR-TRPM7, or siR-TRPV4. *P < 0.05 vs. Control and Cont-siR. The results shown indicate that TRPM7 and TRPV4 are necessary for shear stress-induced increase in [Ca2+]cyt in normal and IPAH-PASMC.

Article Snippet: PASMC were transiently transfected with control siRNA (10 μM, sc-37007; Santa Cruz Biotechnology), or TRPM7 siRNA (10 μM, sc-42662; Santa Cruz Biotechnology) or TRPV4 siRNA (10 μM, sc-64726; Santa Cruz Biotechnology) using Xfect siRNA transfection reagent protocol (Clontech Laboratories).

Techniques: Western Blot

Journal: Disease Models & Mechanisms

Article Title: Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg 2+ -sensitive Socs3a signaling in development and cancer

doi: 10.1242/dmm.004564

Figure Lengend Snippet: The swd locus is allelic with the trpm7 gene. (A) Exocrine pancreas of WT, the trpm7 j124e1 mutant, the swd p75fm / trpm7 j124e1 mutant and the swd p75fm mutant on 5 dpf by immunohistochemistry using anti-Cpa antibodies followed by cross-sectional histological analysis. Staining with DAPI was used to visualize the nuclei. The histological sections are oriented as indicated: d, dorsal; v, ventral; r, right; l, left. (B) Morphometric analysis of exocrine pancreatic epithelial growth (area, in μm 2 , per cell) in the trpm7 j124e1 mutants and WT siblings on 3 and 5 dpf. Each value represents the mean + s.e.m. Statistical analysis was performed using Student’s t -test, with * P <0.05 considered statistically significant. (C) Bright-field images in right lateral view and whole-mount in situ hybridization using anti- trypsin riboprobes. The swd p75fm /+ and trpm7 j124e1 /+ heterozygotes were crossed with each other, and the progeny larvae were analyzed on 5 dpf. The WT, but not mutant, larvae were grown in medium supplemented with PTU, which inhibits skin pigmentation, in order to facilitate visualization of the exocrine-pancreas-expressing trypsin. Note the regions of skin pigmentation (green arrowheads) and trypsin -expressing exocrine pancreas (red arrows) in the mutants as compared to WT.

Article Snippet: PANC-1 and BxPC-3 were grown to 70–80% confluency, trypsinized and resuspended at 10 6 cells in 100 μl of Nucleofector Solution (Amaxa/Lonza) containing 600 nM siRNA directed against human TRPM7 (sc-42662; pre-made and validated by Santa Cruz Biotechnology).

Techniques: Mutagenesis, Immunohistochemistry, Staining, In Situ Hybridization, Expressing

Journal: Disease Models & Mechanisms

Article Title: Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg 2+ -sensitive Socs3a signaling in development and cancer

doi: 10.1242/dmm.004564

Figure Lengend Snippet: The exocrine pancreas of trpm7 b508 mutants is relatively small, and the proliferative defect can be improved by supplementary Mg 2+ or by antisense knockdown of socs3a

Article Snippet: PANC-1 and BxPC-3 were grown to 70–80% confluency, trypsinized and resuspended at 10 6 cells in 100 μl of Nucleofector Solution (Amaxa/Lonza) containing 600 nM siRNA directed against human TRPM7 (sc-42662; pre-made and validated by Santa Cruz Biotechnology).

Techniques:

Journal: Disease Models & Mechanisms

Article Title: Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg 2+ -sensitive Socs3a signaling in development and cancer

doi: 10.1242/dmm.004564

Figure Lengend Snippet: Antisense inhibition of zebrafish trpm7 expression phenocopies the swd mutation. (A) Bright-field images (right lateral view) and whole-mount in situ hybridization using anti- trypsin riboprobes (viewed in the dorsoventral direction) at 96 hpf. Note the region of pigmented skin (green arrows) and trypsin-expressing exocrine pancreas (red arrows) in the trpm7 -ATG-MO-injected larvae as compared with the control. For in situ hybridization, the control-MO-injected embryos were grown in medium supplemented with PTU, which inhibits skin pigmentation, in order to facilitate visualization of the trypsin-expressing exocrine pancreas. This experiment was performed three times, with 25 embryos being injected for each group in each experiment. About 80–90% of larvae in each experiment exhibit the phenotype shown. The residual skin pigmentation is probably due to incomplete knockdown of trpm7 by the MO. (B) Acinar size and morphology revealed by immunohistochemistry using anti-Cpa or anti-cadherin (Cad) antibodies, followed by histological analysis at 96 hpf. Staining with DAPI was used to visualize the nuclei. The histological sections are oriented as indicated: d, dorsal; v, ventral; r, right; l, left. (C) Proportion of exocrine pancreatic epithelia in the S phase of the cell cycle (% BrdU+ nuclei) at 72 hpf. (D) Pancreatic epithelial cell growth (area in μm 2 per cell) by morphometric analysis at 96 hpf. Each value represents the mean + s.d. * P <0.05 indicates statistically significant difference.

Article Snippet: PANC-1 and BxPC-3 were grown to 70–80% confluency, trypsinized and resuspended at 10 6 cells in 100 μl of Nucleofector Solution (Amaxa/Lonza) containing 600 nM siRNA directed against human TRPM7 (sc-42662; pre-made and validated by Santa Cruz Biotechnology).

Techniques: Inhibition, Expressing, Mutagenesis, In Situ Hybridization, Injection, Immunohistochemistry, Staining

Journal: Disease Models & Mechanisms

Article Title: Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg 2+ -sensitive Socs3a signaling in development and cancer

doi: 10.1242/dmm.004564

Figure Lengend Snippet: TRPM7 is overexpressed in human pancreatic adenocarcinoma and required for cellular proliferation. (A) Expression of TRPM7 protein in human pancreatic adenocarcinoma (upper panel) and normal pancreatic tissue (lower panel) by immunohistochemistry using anti-TRPM7 antibodies. The images shown are representative of five tumor specimens and five normal control samples. Scale bars: 100 μm. (B) Relative TRPM7 mRNA levels in human pancreatic adenocarcinoma cell lines by quantitative real-time PCR. Each value represents the mean ratio of each pancreatic cancer cell line to that of H6c7 from triplicate samples, and reproducible results were obtained from two independent experiments. (C) Cell morphology at 24 hours post-transfection with siRNA was examined under an inverted microscope with phase contrast. Red arrows point to multinucleated cells with cytoplasmic vacuoles.

Article Snippet: PANC-1 and BxPC-3 were grown to 70–80% confluency, trypsinized and resuspended at 10 6 cells in 100 μl of Nucleofector Solution (Amaxa/Lonza) containing 600 nM siRNA directed against human TRPM7 (sc-42662; pre-made and validated by Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction, Transfection, Inverted Microscopy

Journal: Disease Models & Mechanisms

Article Title: Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg 2+ -sensitive Socs3a signaling in development and cancer

doi: 10.1242/dmm.004564

Figure Lengend Snippet: TRPM7 controls cellular proliferation by regulating cell-cycle progression and by modulating p21 CDKN1A , cyclin G1 and cyclin B1 expression and Mg 2+ level. (A) PANC-1 cells were treated with siRNA directed against TRPM7 , non-targeting control siRNA, or without siRNA, and then analyzed by MTS assay and counting cells. Proliferation in the anti- TRPM7- siRNA-treated cells is represented as percent of that in the cells treated with non-targeting control siRNA. Each column represents the mean from triplicate samples. Bars represent s.d. * P <0.05 indicates statistical significance. (B) Cell cycle at 72 hours following transfection with siRNA was determined by flow cytometric analysis of DNA content. The proportion of cells in each phase of the cell cycle is indicated. (C) Total RNA was extracted from BxPC-3 and PANC-1 cells at 72 hours following transfection with anti- TRPM7 or non-targeting control siRNA, and analyzed for p21 CDKN1A , cyclin G1 and cyclin B1 mRNA by quantitative real-time PCR. Each column represents the mean from triplicate samples in TRPM7-deficient cells as % of that in control-siRNA-transfected cells. Similar results were obtained from at least two independent experiments. (D) Proliferation assay of PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA, incubated in culture medium with or without added 1 mM MgSO 4 for 72 hours by measuring MTS absorbance or counting viable cells. Bars represent s.d. * P <0.05; NS, not statistically significant.

Article Snippet: PANC-1 and BxPC-3 were grown to 70–80% confluency, trypsinized and resuspended at 10 6 cells in 100 μl of Nucleofector Solution (Amaxa/Lonza) containing 600 nM siRNA directed against human TRPM7 (sc-42662; pre-made and validated by Santa Cruz Biotechnology).

Techniques: Expressing, MTS Assay, Transfection, Real-time Polymerase Chain Reaction, Proliferation Assay, Incubation