Journal: The FASEB Journal

Article Title: KCNH6 is essential for insulin secretion by regulating intracellular ER Ca²⁺ store

doi: 10.1096/fj.202302194rr

Figure Lengend Snippet: FIGURE 4 Altered intracellular Ca2+ stores in β cells. (A) The cytosolic Ca2+ concentration is represented by (F−F0)/(F0−Fb) of Fluo-4, where F0 is baseline fluorescence and Fb is background fluorescence. The calculated AUCs are shown next to the corresponding fluorescence intensity image. (A, B) Thapsigargin (1 μM)- or ionomycin (5 μM)-triggered Ca2+ transients in primary pancreatic β cells from WT (n = 16) and βKO (n = 12) mice in Ca2+-free KRBB solution. (C, D) Glucose (16.7 mM) or KCl (40 mM) stimulated an increase in the intracellular Ca2+ concentration in primary islet β-cells from WT (n = 13) and βKO (n = 15) mice in Ca2+-containing KRBB solution without EGTA. (E) Electrical activity in response to 15 mM glucose recording from WT or βKO β-cell.

Article Snippet: A total of 1 μM thapsigargin (Alomone Labs) or 5 μM ionomycin (Alomone Labs) were added at the indicated times to trigger Ca2+ release from the ER.

Techniques: Concentration Assay, Fluorescence, Activity Assay

Journal: eLife

Article Title: Mechanically stimulated ATP release from murine bone cells is regulated by a balance of injury and repair

doi: 10.7554/eLife.37812

Figure Lengend Snippet: ( A ) Confocal images of CB-OB loaded with quinacrine and MANT-ATP, . ( B ) Changes in quinacrine-loaded CB-OB treated with ionomycin ( left ) or stimulated with micropipette ( right , region of interest in white). ( C ) Vesicular release event in CB-OB ( top ) coincides with a sudden drop in granular quinacrine fluorescence ( bottom ), .( D ) Kinetics of vesicular release from CB-OB stimulated by micropipette at 0 s, n = 37 stimulated cells. ( E–H ) Quinacrine- ( E, F ) or Fura2- ( G, H ) loaded CB-OB were pre-treated (10 min) with vehicle, ionomycin (iono) or NEM or placed in [Ca 2+ ]-depleted physiological solution, and micropipette-stimulated when indicated (+). Vesicular density ( E ) and cumulative release ( F ), secondary responsiveness ( G ) and [Ca 2+ ] response activation rates ( H ) were determined, n = 7–37 primary cells. ( I ) tFSS was applied by replacing 50% media volume n times. ( J, K ) ATP released per cell ( left axis ) or as percent of cellular ATP content ( right axis ) was measured following CB-OB stimulation by tFSS ( J , black dashed line : rational function fit; red dashed line: corresponding asymptote) or after indicated pre-treatments followed by tFSS ( K , 10x media displacements, +), n = 6–8 independent cultures. For , means ± SEM, *significance compared to vehicle ( E–H ), basal ATP release ( J ) or to tFSS-stimulated vehicle ( K ) by ANOVA. Source data for is provided in . 10.7554/eLife.37812.017 Figure 3—source data 1.

Article Snippet: Chemical compound, drug , Ionomycin calcium salt; Iono , Tocris Bioscience , Cat. 1704 , calcium ionophore, 100 μM.

Techniques: Fluorescence, Activation Assay

Journal: eLife

Article Title: Mechanically stimulated ATP release from murine bone cells is regulated by a balance of injury and repair

doi: 10.7554/eLife.37812

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , Ionomycin calcium salt; Iono , Tocris Bioscience , Cat. 1704 , calcium ionophore, 100 μM.

Techniques: In Vivo, Software, Cell Culture, In Vitro, Viability Assay, Staining, Luciferase, ATP Bioluminescent Assay, ALP Assay, Modification

Journal: The Journal of Cell Biology

Article Title: Depolarization induces nociceptor sensitization by Ca V 1.2-mediated PKA-II activation

doi: 10.1083/jcb.202002083

Figure Lengend Snippet: The increase of pRII intensity induced by KCl is independent of NaV, TRPA1, TRPV1, or ionomycin-induced calcium influx . (A) Time course of pRII intensity in lidocaine-pretreated (2, 20, and 200 µM; 10 min) rat DRG neurons stimulated with KCl (40 mM). (B) Dose–response curve of pRII intensities in DRG neurons of WT, TRPA1, TRPV1, and TRPA1/V1 double-knockout mice exposed to KCl (0–80 mM, EC 50 = 10 mM) for 3 min. (C) Calcium imaging (FLIPR Calcium 5 dye) showing calcium influx evoked by ionomycin (2 or 5 µM) followed by KCl (40 mM). Values in are means ± SEM; n = 4; >1,000 neurons/condition. (D) Time course of pRII intensities in DRG neurons stimulated with ionomycin (2 or 5 µM) versus KCl (40 mM). Data in A, B, and D are means ± SEM; n = 3–4 independent experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; *, P < 0.05; **, P < 0.01; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions. Calcium imaging data in C are means ± SEM; n = 4 independent experiments with a total of >500 analyzed neurons/condition.

Article Snippet: FK 506 (50 mM in DMSO), forskolin (10 mM in DMSO), ionomycin calcium salt (10 mM in DMSO), KN-92 (10 mM in DMSO), KN-93 (10 mM in DMSO), [Leu 5 ]-enkephalin (1 mM in 0.1% BSA in dH 2 O), SQ22536 (50 mM in DMSO), and ST 034307 (50 mM in DMSO) were from Tocris.

Techniques: Double Knockout, Imaging

Journal: Oncotarget

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death.

doi: 10.18632/oncotarget.4487

Figure Lengend Snippet: Figure 4: Ionomycin enhanced CGK733-induced PERK/CHOP activation and vesicular calcium sequestration. A. The expression of PERK and CHOP were detected by Western blotting after cells were treated with CGK733 for 6 h in the presence or absence of 1 μM of ionomycin. B. MTS viability assays were performed after cells were treated with CGK733 for 24 h in the presence or absence of 1 μM of ionomycin. C. The vesicles were observed under a microscope after PK45-p and PK59 cells were treated with 10 μM of CGK733 in the presence or absence of 1 μM of ionomycin for 12 h and 9 h, respectively. D. Cal-520 fluorescence combined with bright field microscopy was performed after cells were exposed to 20 μM of CGK733 for 12 h in the presence or absence of 1 μM of ionomycin. Bars, SD; Black arrows indicate the observed vesicles; red arrows indicate the co-localization of calcium ions with the vesicles.

Article Snippet: Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany.

Techniques: Activation Assay, Expressing, Western Blot, Microscopy, Fluorescence