Journal: iScience

Article Title: Type 2 diabetes is associated with increased circulating levels of 3-hydroxydecanoate activating GPR84 and neutrophil migration

doi: 10.1016/j.isci.2022.105683

Figure Lengend Snippet: 3-Hydroxydecanoate signals through Gα i , Gα q , and GPR84 (A) Baseline normalized cell index (CI) of cells stimulated with 3-hydroxydecanoate (3-OH-C10) or the positive control compound, zaprinast, in the xCELLigence assay (N = 3). (B–D) Maximum baseline normalized CI of cells stimulated with 3-hydroxydecanoate after pre-incubation with (B) the Gα i inhibitor PTX (200 ng/mL) for 16 h (N = 3–4), (C) the Gα q inhibitor YM-254890 (2 μM) for 15 min (N = 5–7), or (D) the β-arrestin inhibitor barbadin (1 μM) for 15 min (N = 3–4). (E and F) PRESTO-Tango luciferase assay in HTLA cells transfected with human GPR84. (E) Transfected cells were stimulated with 3-hydroxydecanoate, the endogenous GPR84 agonist decanoate (capric acid, C10), the GPR84 reference agonist embelin or the GPR84 antagonist AR505962 (N = 4–5). (F) Transfected cells were pre-incubated with the GPR84 antagonist AR505962, followed by stimulation with sub-maximal concentrations of 3-hydroxydecanoate, decanoate, or embelin (N = 3–4). (G) PRESTO-Tango assay in HTLA cells transfected with human GPR109B (HCA3). The cells were stimulated with 3-hydroxydecanoate, the endogenous GPR109B agonist 2-hydroxyoctanoate (2-OH-C8), or the GPR109B reference agonist AR231783. N = 3. Data are shown as mean ± SEM The logEC50 values were calculated in GraphPad Prism using nonlinear regression.∗p < 0.05, ∗∗p < 0.01. p values were determined by one-way ANOVA followed by Tukey’s multiple comparison test (B–D). See also Figure S3 .

Article Snippet: For antagonist mode, the cells were pre-incubated with 2 μM YM-254890 (FUJIFILM Wako Chemicals #257–00631) or 10 μM barbadin (Toronto Research Chemicals #B118250) for 15 min with readings performed every 15 s; followed by agonist stimulation as described.

Techniques: Positive Control, Incubation, Luciferase, Transfection

Journal: Cancers

Article Title: Dissecting the Mechanism of Action of Spiperone—A Candidate for Drug Repurposing for Colorectal Cancer

doi: 10.3390/cancers14030776

Figure Lengend Snippet: Spiperone induces Ca 2+ and PLC-dependent cell death. Effect of co-treatment with spiperone and 1 μmol/L BAPTA-AM, 1 μmol/L EGTA, 2 μmol/L U73122, and 10 μmol/L 2APB in HCT116 cells. After 30 min of pretreatment, cells were co-treated with 10 μmol/L spiperone or vehicle for 24 h ( a ). Effect of co-treatment with spiperone and 1 μmol/L BAPTA-AM or 2 μmol/L U73122 in SW480 ( b ) and HCT8 cells ( c ). After 30 min of pretreatment, 10 μmol/L spiperone or vehicle were added, and co-treatment was maintained for 24 h. Effect of PLC silencing on spiperone-induced cell death. HCT116-silenced cells were treated for 48 with 5 μmol/L spiperone ( d ). Graphs displaying cell viability as the percentage of viable cells. Data show the mean ± SD of at least three independent experiments performed in triplicate. *: Student’s t -test p < 0.05; **: Student’s t -test p < 0.01; ***: Student’s t -test p < 0.001; ****: Student’s t -test p < 0.0001.

Article Snippet: Subsequently, all of the samples were resuspended for 30 min in 2 mmol/L CaCl 2 KRB, 2 mmol/L EGTA KRB, or 2 mmol/L EGTA KRB with 10 μmol/L U-73122 (Sigma-Aldrich, Saint Louis, MO, USA), 10 μmol/L U-73443, 50 μmol/L 2APB, 50 μmol/L genistein, 10, 20, and 50 μmol/L regorafenib, 10 μmol/L CID-1067700, 10 μmol/L gallein, 10 μmol/L YM-254890 (Cayman Chemicals), or 50 μmol/L suramin (Cayman Chemicals, Ann Arbor, MI, USA).

Techniques:

Journal: Pflugers Archiv

Article Title: Functional expression of the proton sensors ASIC1a, TMEM206, and OGR1 together with BK Ca channels is associated with cell volume changes and cell death under strongly acidic conditions in DAOY medulloblastoma cells

doi: 10.1007/s00424-024-02964-7

Figure Lengend Snippet: DAOY cells express functional OGR1. (a) Left, representative images of Ca 2+ -imaging experiments. Middle, mean ± SD of all Ca 2+ -imaging experiments of DAOY cells stimulated with pH 6.0 and pH 5.0. Right, summary data normalized to the response to ionomycin (iono) ( n = 54). (b) Left, representative images of Ca 2+ -imaging experiments. Middle, mean ± SD of all Ca 2+ -imaging experiments of DAOY cells stimulated with a co-application of 1 μM YM-254890 (YM) and pH 5.0. YM was preincubated for 30 min. Right, summary data normalized to background (bgd) ( n = 108). (c) mRNA expression of OGR1 in DAOY and UW228 cells cultured with a pH 7.4 medium (ctrl) and in DAOY cells cultured as MS for 7 days. qPCR data was normalized to HPRT1 expression levels ( n = 3). Iono was used as a positive control for all Ca 2+ -imaging experiments. Summary data are shown as box plots (a, b) or as mean ± SD (c). Statistical analysis was performed with Wilcoxon signed-ranked tests (a, b) and a repeated measures one-way ANOVA (c). *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001

Article Snippet: PcTx1 and IbTx (Smartox Biotechnology, Sainte-Égrève, France), YM-254890 (Biozol, Eching, Germany), DIDS (Sigma-Aldrich, St. Louis, MI, USA), bepridil hydrochloride (MedChemExpress, Monmouth Junction, New Jersey, United States), NS-11021 (Alomone Labs, Jerusalem, Israel), and pregnenolone sulfate (Sigma-Aldrich) were purchased in research quality.

Techniques: Functional Assay, Imaging, Expressing, Cell Culture, Positive Control