Journal: Cardiovascular Research

Article Title: Contractility measurements for cardiotoxicity screening with ventricular myocardial slices of pigs

doi: 10.1093/cvr/cvad141

Figure Lengend Snippet: Screening test with drugs from CiPA list with known interactions and risks performed in three different laboratories ( N = 3–5, n = 6–10). The effects of bepridil, cisapride, disopyramide, ibutilide, and risperidone (red) as well as negative controls DMSO and acetylsalicylic acid (black) were compared to time-matched controls treated only with PBS (grey). ( A ) Kaplan–Meier curve displaying the pacing capability of slices. Statistical analysis by Fisher’s exact test. ( B–H ) Mean and SEM of effects compared to PBS with a two-way ANOVA with Dunnett’s multiple comparison test on the normalized pacing threshold ( B ), normalized force ( C ), FFR ( D ), normalized contraction duration ( E ), CDFR ( F ), normalized ERP ( G ), and changes in diastolic force ( H ). Absolute values of each slice were normalized to values before adding the drug or solvent. Exact n and P values for each condition are given in , .

Article Snippet: Drugs from the CiPA list were added at five different concentration steps into 3–4 mL from the following stocks: bepridil hydrochloride (Tocris Bioscience, UK) 10 mM dissolved in DMSO; ibutilide hemifumarate (Sigma-Aldrich, USA) 10 μM dissolved in H 2 O; disopyramide phosphate (Sigma-Aldrich, USA) 10 mM dissolved in H 2 O; cisapride monohydrate 100 μM dissolved in DMSO; risperidone (Sigma-Aldrich, USA) 1 mM dissolved in DMSO; and acetylsalicylic acid (Sigma-Aldrich, USA) 100 μM dissolved in H 2 O.

Techniques: Comparison, Solvent

Journal: Frontiers in Physiology

Article Title: Store-operated calcium entry and the localization of STIM1 and Orai1 proteins in isolated mouse sinoatrial node cells

doi: 10.3389/fphys.2015.00069

Figure Lengend Snippet: List of antibody used in the study .

Article Snippet: ORAI3 , ProSci (4117) , Polyclone , Rabbit , 1:200 , 1:1000.

Techniques: Staining, Western Blot, Transduction

Journal: Frontiers in Physiology

Article Title: Store-operated calcium entry and the localization of STIM1 and Orai1 proteins in isolated mouse sinoatrial node cells

doi: 10.3389/fphys.2015.00069

Figure Lengend Snippet: Expression of STIM and Orai molecules in mouse cardiac myocytes. (A) Total RNA was isolated from pacemaker cells (SAN), atrial myocytes (Atrium), ventricular myocytes (Ventricle) and spleen. Transcripts of Stim1, Stim2, Orai1, Orai2, and Orai3 in cardiac myocytes were compared with the spleen as positive control. The housekeeping gene Hypoxanthine–Guanine Phosphoribosyltransferase (HPRT1) was used as an internal control. (B) Protein levels of STIM1, STIM2, Orai1, and Orai3 were examined in total protein extracts from SAN region (SAN), atrium (Atrium), ventricle (Ventricle) and spleen and determined by Western Blot analysis. The nuclear protein histone was used as loading control. (C) Bar graph showing quantitative western blot analysis of STIM1 protein levels in spleen and heart with heart samples normalized to spleen as 1 ( n = 3 gels, P < 0.05, ANOVA). (D) Confocal images of an isolated pacemaker cell shows expression of HCN4 (green in color), a positive marker for pacemaker cells. The cell also positively stained with anti-Orai1 antibody (red in color). The nucleus is stained blue with DAPI. A merged image on the left shows the co-localization of HCN4 and Orai1 along the surface membrane (color in yellow). (E) Representative confocal images of an isolated pacemaker cell labeled with STIM1 (green in color) and DAPI (blue in color). When perfused with normal Ca 2+ containing solution, positive STIM1 staining was distributed both intracellularly and in the cell periphery as showed in the top panel. When perfused with Ca 2+ -free solution containing thapsigargin, STIM1 peripheral localization was enhanced, as shown in the bottom panel.

Article Snippet: ORAI3 , ProSci (4117) , Polyclone , Rabbit , 1:200 , 1:1000.

Techniques: Expressing, Isolation, Positive Control, Control, Western Blot, Marker, Staining, Membrane, Labeling

Journal: British Journal of Pharmacology

Article Title: Blockade of the forward Na + /Ca 2+ exchanger suppresses the growth of glioblastoma cells through Ca 2+ ‐mediated cell death

doi: 10.1111/bph.14692

Figure Lengend Snippet: Recording and blockade of the Na + /Ca 2+ exchanger (NCX) currents in glioblastoma cells. (a) Human glioblastoma U87 cells were held at −50 mV; the membrane currents associated with NCX were recorded by a voltage ramp from +60 to −120 mV. Ni 2+ (10 mM) was used to block the NCX currents. (b, c) NCX currents recorded before and after application of bepridil (0.1–100 μM) or KB‐R7943 (0.1–100 μM). (d–f) The currents recorded before and after application of the reverse NCX blockers SEA0400 (0.1–100 μM), SN‐6 (0.1–30 μM), and YM244769 (0.1–30 μM). The reverse NCX current was measured at +50 mV, and forward NCX current was measured at −110 mV

Article Snippet: [1,2‐Bis(2‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA‐AM)] (Cat# 2787), YM‐244769 (Cat# 4544), KB‐R7943 (Cat# 1244), SN‐6 (Cat# 2184), SEA0400 (Cat# 6164), and bepridil (Cat# 4117) were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Membrane, Blocking Assay

Journal: British Journal of Pharmacology

Article Title: Blockade of the forward Na + /Ca 2+ exchanger suppresses the growth of glioblastoma cells through Ca 2+ ‐mediated cell death

doi: 10.1111/bph.14692

Figure Lengend Snippet: Effect of the Na + /Ca 2+ exchanger (NCX) blockers on viability of human glioblastoma cell lines. (a, b) Viability of adult glioblastoma cell lines (U87 and U251) after exposure to NCX blockers bepridil, KB‐R7943, SEA0400, SN‐6, or YM244769 at 0–100 μM for 72 hr. n = 5 independent tests per group. (c) Viability of paediatric glioblastoma cell line SF188 after 72‐hr incubation with the same NCX blockers at 0–100 μM. n = 5 independent tests per group. (d) For U87, U251, and SF188 cell lines, concentration of the NCX blockers required to produce half maximal inhibition (IC 50 : μM, mean ± SEM) of cell viability. IC 50 was obtained by fitting the normalized concentration–response relationship to the equation: V / V 0 = 1/{1 + [C/IC 50 ] n } , where V 0 and V are the cell viability measured in control and in the presence of a blocker, C is the concentration of the blocker, and n is the Hill coefficient. n = 5 independent cultures in each group

Article Snippet: [1,2‐Bis(2‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA‐AM)] (Cat# 2787), YM‐244769 (Cat# 4544), KB‐R7943 (Cat# 1244), SN‐6 (Cat# 2184), SEA0400 (Cat# 6164), and bepridil (Cat# 4117) were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Incubation, Concentration Assay, Inhibition, Control

Journal: British Journal of Pharmacology

Article Title: Blockade of the forward Na + /Ca 2+ exchanger suppresses the growth of glioblastoma cells through Ca 2+ ‐mediated cell death

doi: 10.1111/bph.14692

Figure Lengend Snippet: Effect of bepridil and KB‐R7943 on the level of [Ca 2+ ] i measured by Ca 2+ imaging. (a) Flua‐4 AM‐loaded U87 cells were imaged and perfused with bepridil (25 or 50 μM) in the HEPES‐buffered solution containing 2‐mM Ca 2+ . (b) Ca 2+ imaging of U87 cells before and after perfusion with KB‐R7943 (25 or 50 μM). (c) In the Ca 2+ ‐free solution, Ca 2+ imaging of U87 cells before and after perfusion with bepridil or KB‐R7943. (d) Ca 2+ imaging before and after perfusion with bepridil or KB‐R7943, followed by application of 5‐μM thapsigargin. (e) U87 cells were pretreated with a Ca 2+ chelator BAPTA‐AM (20 μM) for 2 hr and then imaged and perfused with bepridil or KB‐R7943. The traces are representative of three separate experiments, and n = 12–18 cells were imaged per experiment

Article Snippet: [1,2‐Bis(2‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA‐AM)] (Cat# 2787), YM‐244769 (Cat# 4544), KB‐R7943 (Cat# 1244), SN‐6 (Cat# 2184), SEA0400 (Cat# 6164), and bepridil (Cat# 4117) were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Imaging

Journal: British Journal of Pharmacology

Article Title: Blockade of the forward Na + /Ca 2+ exchanger suppresses the growth of glioblastoma cells through Ca 2+ ‐mediated cell death

doi: 10.1111/bph.14692

Figure Lengend Snippet: Impact of bepridil on human astrocytes (HA) and the Na + /Ca 2+ exchanger (NCX) isoforms in each cell line. (a) Viability of U87, U251, and SF188 cells and HA after exposure to bepridil (25 μM, 48 hr); n = 5 independent tests in each group. (b) The bands of NCX1, NCX2, and NCX3 isoforms detected by Western blot analysis in the sample of HA and glioblastoma cell lines U87, U118, A172, U251, and SF188. (c) Representative recording of the NCX currents in HA and glioblastoma cell lines. (d) Viability of U87, U251, and SF188 cells and HA after exposure to KB‐R7943 (25 μM, 48 hr). n = 5 independent tests in each group

Article Snippet: [1,2‐Bis(2‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA‐AM)] (Cat# 2787), YM‐244769 (Cat# 4544), KB‐R7943 (Cat# 1244), SN‐6 (Cat# 2184), SEA0400 (Cat# 6164), and bepridil (Cat# 4117) were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Western Blot

Journal: British Journal of Pharmacology

Article Title: Blockade of the forward Na + /Ca 2+ exchanger suppresses the growth of glioblastoma cells through Ca 2+ ‐mediated cell death

doi: 10.1111/bph.14692

Figure Lengend Snippet: Concentration of compounds required to produce half‐maximal inhibition (IC 50 : μM) of the currents related to the reverse and forward NCX operation

Article Snippet: [1,2‐Bis(2‐aminophenoxy)ethane‐ N , N , N ′, N ′‐tetraacetic acid (BAPTA‐AM)] (Cat# 2787), YM‐244769 (Cat# 4544), KB‐R7943 (Cat# 1244), SN‐6 (Cat# 2184), SEA0400 (Cat# 6164), and bepridil (Cat# 4117) were purchased from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Concentration Assay, Inhibition, Blocking Assay