Journal: Frontiers in Cardiovascular Medicine

Article Title: Signaling and structures underpinning conducted vasodilation in human and porcine intramyocardial coronary arteries

doi: 10.3389/fcvm.2022.980628

Figure Lengend Snippet: K + channel-mediated vasodilation underlies conducted dilation of human and porcine RA-IMCAs. (A) Schematic of experimental setup with direction of flow of bradykinin (BK) indicated by carboxyfluorescein (CF). Micrographs showing an isolated, cannulated and pressurized human (B ) and porcine (C) artery. Focal application of BK (and CF, green) to the downstream end of the artery against the direction of superfusion flow caused local and conducted dilation in both human (D) and porcine (E) arteries. The corresponding time course of responses are shown in (F,G) , and , . A bolus of bradykinin was delivered at the point indicated by the arrow, and simultaneous inner diameter measured locally (0 μm) and up to 1,000 μm upstream, positions indicated by arrows in (D,E) . The same human artery used for K Ca 3.1 immunolabel in . Summary graphs show that compared to control ( n = 9, 14) neither L-NAME (100 μM, n = 3, 6) nor Ba 2+ (30 μM, n = 3, 3) affected local or conducted dilation, whereas depolarization to 45 mM KCl abolished conducted dilation ( n = 3, 6) in human (H) and porcine (I) arteries, respectively. Non-parametric mixed effects analysis with Sidak's multiple comparison test; * P < 0.05 vs. control.

Article Snippet: Primary antibodies were as follows: 1:100 rabbit polyclonal anti-rat K Ca 3.1 (aa 350–363; Alomone Laboratories, APC-064); 1:100 mouse monoclonal anti-human K Ca 3.1 (third extracellular loop; Alomone Laboratories, ALM-051); and 1:100 rabbit polyclonal anti-human K Ca 2.3 (aa 2–21, Alomone Laboratories, APC-025).

Techniques: Isolation, Immunolabeling

Journal: Frontiers in Cardiovascular Medicine

Article Title: Signaling and structures underpinning conducted vasodilation in human and porcine intramyocardial coronary arteries

doi: 10.3389/fcvm.2022.980628

Figure Lengend Snippet: K + channel expression and hyperpolarization to bradykinin in human and porcine IMCAs. Confocal micrographs of immunolabelling for K Ca 3.1 and K Ca 2.3 in isolated, cannulated and pressurized human (A) and porcine (B) RA-IMCAs with myogenic tone and full dilation to BK. Punctate and diffuse K Ca 3.1 label was evident in the ECs of human and porcine arteries, whereas K Ca 2.3 was less clear in human IMCAs, and highly expressed at EC borders of porcine IMCAs (yellow arrowheads). The elastin was dense in human arteries, with the internal elastic lamina (IEL) seen as longitudinal strings in the porcine arteries. Representative of at least 3 arteries for each label; asterisks indicate corresponding nuclei in upper and lower panels. The pink dashed line in the schematic represents the focal plane. (C) The schematic indicates a sharp microelectrode impaled into a SMC of a porcine RA-IMCA mounted for isometric tension recording. Under control conditions the thromboxane mimetic U46619 (0.6 μM) depolarized and contracted arteries, and BK (10 nM) caused hyperpolarization and relaxation (C) , summarized in (D) . (E) Addition of L-NAME depolarized and contracted porcine left ventricular (LV)-IMCAs. Under these conditions BK (0.1 nM to 100 nM) repolarized and relaxed the artery. Addition of 100 nM acetylcholine (ACh) depolarized and contracted the artery. The asterisk indicates when the electrode came out of the cell. The RMP and tension prior to the addition of L-NAME (100 μM) are indicated by dashed lines. Drugs were added to a static bath at the arrows. RMP, resting membrane potential.

Article Snippet: Primary antibodies were as follows: 1:100 rabbit polyclonal anti-rat K Ca 3.1 (aa 350–363; Alomone Laboratories, APC-064); 1:100 mouse monoclonal anti-human K Ca 3.1 (third extracellular loop; Alomone Laboratories, ALM-051); and 1:100 rabbit polyclonal anti-human K Ca 2.3 (aa 2–21, Alomone Laboratories, APC-025).

Techniques: Expressing, Isolation

Journal: PLoS ONE

Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

doi: 10.1371/journal.pone.0154471

Figure Lengend Snippet: (A-D) SK4 IHC in fours subtypes of breast cancer tissues including Luminal A (A), Luminal B (B), HER2 (C), and TNBC (D). Scale bars, 50 μm. (E) Immunoblotting of SK4 and E-cadherin in breast cancer tissues (BC1 and BC2) and non-tumor breast tissues (Non-Tumor1 and Non-Tumor2).

Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

doi: 10.1371/journal.pone.0154471

Figure Lengend Snippet: Comparison of SK4 expression in tumor subtypes for 50 patients using Fisher’s exact test.

Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

doi: 10.1371/journal.pone.0154471

Figure Lengend Snippet: (A) Immunoblotting of SK4 and EMT-related proteins (E-cadherin and Vimentin) in breast cancer cell lines. (B) Comparison of SK4 mRNA expression in 4 breast cancer cell lines as determined by real-time PCR; n = 3. (C) Immunoblotting of ER protein in MDA-MB-468, MDA-MB-231 and T47D cells. (D-G) Immunostaining of SK4 (red) and nuclear marker DAPI (blue) in MDA-MB-231 (D), MDA-MB-468 (E), MCF-7 (F) and T47D (G) cells. Scale bars, 50 μm. (H, I) Whole-cell recording of MDA-MB-231 cells before (H) and after (I) 5-μM TRAM-34 treatment. (J, K) With (J) or without (K) 350 nM free Ca 2+ in the pipette solution, the voltage-current density curves show the currents changes before (a) and after (b) TRAM-34 treatment. The currents were evoked by step voltage ranging from -100 mV to +100 mV in steps of 10 mV every 100 ms. Dunnett’s Multiple Comparison Test was applied in comparison, ** p <0.01.

Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Marker, Transferring

Journal: PLoS ONE

Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

doi: 10.1371/journal.pone.0154471

Figure Lengend Snippet: A negative control siRNA (N.C.) and 3 SK4-specific siRNAs (Si-1, Si-2 and Si-3) were transfected into MDA-MB-231 cells, and 20 μM TRAM-34 was added to the TRAM-34-treated group to inhibit SK4 channels. (A, B) Knockdown of SK4 by siRNA was confirmed using immunoblotting and real-time PCR; n = 3. (C, D) The images and bar of the transwell migration assay indicate that the counts of migrated cells in SK4 siRNA (Si-SK4)- and TRAM-34-treated group were significantly less than those of the control (CTL). Scale bars, 50 μm; n = 4. (E, F) The images and bar of the wound-healing assay. The wound-healing rate represents the distance migrated by cells at certain time divided by the wound distance at 0 h. Scale bars, 100 μm; n = 3. The data are presented as the mean ± SD, Dunnett’s Multiple Comparison Test was applied in (B) and (D), and unpaired t test in (F). ** p <0.01, *** p <0.001.

Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

Techniques: Negative Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Transwell Migration Assay, Wound Healing Assay

Journal: PLoS ONE

Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

doi: 10.1371/journal.pone.0154471

Figure Lengend Snippet: (A) Phase contrast images of MDA-231 and T47D cells treated with (E+b) or without (CTL) EGF/bFGF for 1 day, 3 days and 5 days. Scale bars, 100 μm. (B, C) The EGF/bFGF-induced EMT of MDA-231 cells was confirmed using immunoblotting and real-time PCR of EMT markers (Vimentin, Snail1 and Slug), and the SK4 mRNA level increased after EMT. (D) Immunoblotting of EMT-related proteins (Vimentin and Snail1) was performed 72 h after MDA-231 cells were transfected with negative control siRNA (N.C.) or SK4-specific siRNA (Si-SK4); cells that did not undergo transfection served as a control (CTL). The data are presented as the mean ± SD, and paired t test was applied in comparison. n = 3; * p <0.05.

Article Snippet: Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Transfection, Negative Control

Journal:

Article Title: Both Laminin and Schwann cell Dystroglycan are necessary for proper clustering of Sodium Channels at Nodes of Ranvier.

doi: 10.1523/JNEUROSCI.2068-05.2005

Figure Lengend Snippet: Antibodies

Article Snippet: Secondary antibodies included the following: fluorescein isothiocyanate (FITC)- or tetramethylrhodamine isothiocyanate (TRITC)-conjugated donkey anti-rabbit IgG (1:100); FITC-conjugated donkey anti-mouse IgG (1:150) and FITC-conjugated goat anti-rat IgG were from Jackson Immuno Research. table ft1 table-wrap mode="anchored" t5 Antigen Antibody Species Clone/name Dilution Source α−dystroglycan mAb mouse IIH6 1:100 Kevin Campbell β−dystroglycan mAb mouse 43DAG1/8D5 1:50 NovoCastra Laboratories Dystrophin Dp116 mAb mouse MANDRA1 1:100 SIGMA DRP2 pAb rabbit 2164 1:100 Peter Brophy Ezrin pAb rabbit 1:30 Upstate ERM phosphorylated pAb rabbit 1:50 Cell Signaling Caspr mAb mouse 1:100 Elior Peles Caspr pAb rabbit 1:600 Elior Peles Lamininα1 mAb rat 317 1:2 Lydia Sorokin Lamininα2 mAb rat 4H8/2 1:100 Alexis Lamininα4 pAb rabbit 1:100 Jeffrey Miner Lamininα5 pAb rabbit 1:400 Lydia Sorokin Lamininβ2 pAb rabbit 1117 1:50 Rupert Timpl Lamininγ1 mAb rat A5 1:50 Chemicon MAG mAb mouse 1:50 Boehringer Mannheim Neurofascin 155+186 pAb rabbit 1:1000 Peter Brophy Potassium channel Kv 1.1 pAb rabbit 1:100 Alomone Labs Radixin pAb rabbit 92-4 1:100 Heinz Furthmayr Sodium channel Nav 1.6 pAb rabbit 1:200 James Trimmer Sodium channel Nav 1.2 mAb mouse 1:100 Upstate Sodium channel Pan Nav mAb mouse K58/35 1:100 Sigma Utrophin mAb mouse DRP3/20C5 1:50 NovoCastra Laboratories Open in a separate window Antibodies

Techniques: