Journal: SLAS discovery : advancing life sciences R & D

Article Title: Development of a Novel Cell-Based Assay System for High-Throughput Screening of Compounds Acting on Background Two-Pore Domain K + Channels.

doi: 10.1177/2472555219829745

Figure Lengend Snippet: Figure 6. Randomized blind test for the evaluation of TASK-3 modulators using the new cell-based assay. (A) Five test compounds were assigned numbers (T 1–5) at random. Their evaluation as TASK-3 modulators at a concentration of 1 μM was performed by a blind test using the automated procedure and a 96-well plate. The data expressed as a heat map for this trial are shown. The number in each compartment indicates cell viability, taking the control as unity (1.0). Red and green colors indicate annihilation and higher viability than the control, respectively. (B) The effects of T1–T5 on the cell viability measured in A are summarized. (C) The compounds used in this blind test are listed. T1: ML365, T2: NS309, T3: PK-THPP, T4: losartan, T5: atropine. Note that ML365 and PK-THPP show significant inhibitory effects on cell viability, based on their blocking on TASK-3.

Article Snippet: NS309 was obtained from Alomone Labs (Jerusalem, Israel).

Techniques: Cell Based Assay, Concentration Assay, Control, Blocking Assay

Journal: Scientific Reports

Article Title: KCa3.1 channels regulate the tumor infiltration of functionally competent NK cells in head and neck cancer

doi: 10.1038/s41598-025-20101-x

Figure Lengend Snippet: KCa3.1 channels regulate NK cell chemotaxis, cytokine release and cytotoxicity. ( A ) Representative KCa3.1 current traces in activated NK cells from a healthy individual before and after exposure to the selective KCa3.1 blocker TRAM-34 (200 nM). KCa3.1 activity was elicited by depolarizing ramp-pulses from − 120 mV to + 50 mV (holding potential: −70 mV) in whole-cell configuration with 1 µM free Ca 2+ in the pipette. ( B ) Average KCa3.1 activity (reported as conductance) and ( C ) average cell capacitance recorded in 17 resting NK cells and 14 activated NK cells from 4 HDs. ( D ) Single-cell trajectories of a representative experiment of NK cells migrating towards a CXCL10 gradient (green; left) or a combination gradient of CXCL10 and 500 nM TRAM-34 (red; right). Trajectories of 15 cells are shown for each experiment, and the starting point of each cell is artificially set to the same origin. The red triangles represent Y-COM. ( E ) Y-COM, ( F ) Forward migration index-Y (FMI Y ), i.e., the directed active cell movement towards the Y-axis, and ( G ) Velocity of migrating cells measured in activated NK cells migrating along a CXCL10 gradient, or a combination gradient of CXCL10 and TRAM-34 ( n = 5 HDs). ( H ) Y-COM values calculated for activated NK cells migrating towards a CXCL10 gradient with or without 1µM NS309 (a KCa3.1 activator) preincubation in healthy donors ( n = 4 donors). ( I ) Multiplex cytokine release assay showing fold change in the abundance of individual proteins in activated primary NK cells that were treated with either 500 nM TRAM-34 (left) or 1 µM NS309 (right). Vehicle treated cells were used as controls. The abundance of the individual analyte represented in the bars is shown as relative to that in controls (dotted line). Data were measured in n = 5 HDs (the same donors were used for both treatments). (J) Bar graphs showing mean fluorescence intensities (MFI) of caspase 3/7 fluorescence, indicating cell death (left) and Cell Tracker Dye, representing the abundance of NK cells (right) in Cal27 spheroids co-cultured with activated HD NK cells that were treated with either 1 µM NS309 or 500 nM TRAM-34. Vehicle-treated cells served as controls. The MFIs for the NS309 and TRAM-34 groups were normalized to the MFI of the control group. Data were collected from four HDs. (K) Effects of KCa3.1 inhibition and activation on NK cell function (summary of results in Panels D-J). Data for (B , C) were analyzed by unpaired Student’s t-test, for (E-G) by paired Student’s t-test. Data in (H , I) were analyzed by Wilcoxon signed-rank test. Bars represent mean ± SD, and each symbol represents an individual donor. In (I) significance denoted by (*) for P ≤ 0.05 and (#) for P ≤ 0.001. Data in (J) were analyzed with one-way ANOVA ( P = 0.006 for caspase MFI, and P < 0.001 for Cell Tracker MFI), posthoc testing (significant P values shown in graphs) was performed by Tukey’s test.

Article Snippet: DMEM and rat tail collagen I were purchased from Corning Inc., NS309 and ShK-Dap22 were purchased from Tocris Bioscience, and CXCL10 (cat#266-IP/CF) was obtained from R&D Systems.

Techniques: Chemotaxis Assay, Activity Assay, Transferring, Migration, Multiplex Assay, Release Assay, Fluorescence, Cell Culture, Control, Inhibition, Activation Assay, Cell Function Assay

Journal: Scientific Reports

Article Title: KCa3.1 channels regulate the tumor infiltration of functionally competent NK cells in head and neck cancer

doi: 10.1038/s41598-025-20101-x

Figure Lengend Snippet: KCa3.1 channels regulate HNSCC NK cell chemotaxis and the response to adenosine. (A) Single-cell trajectories of a representative experiment of activated NK cells migrating in the absence of chemokine (left), towards a CXCL10 gradient (middle, green), or a combination gradient of CXCL10 and TRAM-34 (right, red). Trajectories of 15 cells are shown for each experiment, and the starting point of each cell is artificially set to the same origin. The red triangles represent the Y-center of mass (Y-COM). (B) Y-COM values calculated for HNSCC NK cells migrating along a CXCL10 gradient, or a combination gradient of CXCL10 and TRAM-34 ( n = 5 HNSCC patients). (C) Single-cell trajectories of a representative experiment of activated NK cells migrating towards either a CXCL10 gradient (left, green) or a mixed gradient of CXCL10 and 10 µM adenosine (ADO) (red, middle), or a mixed gradient of CXCL10 and ADO (red, right). The right panel shows cells migrating in a CXCL10/ADO gradient after pretreatment with 1µM NS309. (D) Y-COM values calculated for HNSCC NK cells migrating along a CXCL10 gradient, or a combination gradient of CXCL10 with ADO with or without preincubation with NS309 ( n = 3 HNSCC patients). (E) Schematic of effects of KCa3.1 modulation on NK cell chemotaxis in HNSCC. Data were analyzed by paired Student’s t-test for (B) , and repeated measures one-way analysis of variance ( P = 0.004) with Grisser-Greenhouse correction for (D) . Post-hoc testing to assess pairwise comparisons in (D) was done by Tukey’s multiple comparison test.

Article Snippet: DMEM and rat tail collagen I were purchased from Corning Inc., NS309 and ShK-Dap22 were purchased from Tocris Bioscience, and CXCL10 (cat#266-IP/CF) was obtained from R&D Systems.

Techniques: Chemotaxis Assay, Comparison

Journal: Scientific Reports

Article Title: KCa3.1 channels regulate the tumor infiltration of functionally competent NK cells in head and neck cancer

doi: 10.1038/s41598-025-20101-x

Figure Lengend Snippet: KCa3.1 does not regulate activation in HNSCC cancer cell proliferation. ( A ) KCNN4 (the gene encoding KCa3.1) mRNA levels were quantified reverse transcription quantitative polymerase chain reaction (RT-qPCR). Data shown are fold change in KCNN4 expression in resting and activated primary CD8 + T cells (left), HNSCC cell lines (middle), and HNSCC patient tumor biopsies (right). The data are normalized to the KCNN4 expression levels in activated CD8 + T cells. Each sample was run in quadruplicate. 18 S rRNA was used as the housekeeping gene. The bars represent mean ± SD, and each symbol corresponds to an individual experiment. ( B ) Effect of activation of KCa3.1 channels by NS309 on the proliferation of HNSCC cell over various time points up to 72 h. Proliferation was measured at 24, 48 and 72 h in Cal27 (blue line) and UMSCC (red line) HNSCC cell lines treated with 1, 5 and 10 mM NS309 using a colorimetric cell proliferation assay. Data are shown as percent change in proliferation of the HNSCC cells for the different NS309 concentrations as compared to untreated controls. Data in (A) were analyzed by unpaired student’s t-test by comparing the KCNN4 abundance in the individual HNSCC cell lines and tumor biopsies and resting CD8 + T cells to activated CD8 + T cells. Data in (B) were analyzed by one-way analysis of variance (ANOVA), and the P-values for the analysis of variance are shown in blue for the Cal27 cells and in red for the UMSCC cells. (P = 0.065 and P = 0.088 for 24h Cal27 and UMSCC respectively, P = 0.052 and P = 0.708 for 48 h Cal27 and UMSCC respectively, and P = 0.089 and P = 0.7577 for 72 h Cal27 and UMSCC respectively).

Article Snippet: DMEM and rat tail collagen I were purchased from Corning Inc., NS309 and ShK-Dap22 were purchased from Tocris Bioscience, and CXCL10 (cat#266-IP/CF) was obtained from R&D Systems.

Techniques: Activation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Proliferation Assay

Journal: British Journal of Pharmacology

Article Title: A new negative allosteric modulator, AP14145, for the study of small conductance calcium‐activated potassium (K Ca 2) channels

doi: 10.1111/bph.14043

Figure Lengend Snippet: Chemical structures of AP14145 (left) and NS8593 (right).

Article Snippet: NS8593 and NS 309 was supplied by NeuroSearch A/S, Ballerup, Denmark.

Techniques:

Journal: British Journal of Pharmacology

Article Title: A new negative allosteric modulator, AP14145, for the study of small conductance calcium‐activated potassium (K Ca 2) channels

doi: 10.1111/bph.14043

Figure Lengend Snippet: Bar graph depicting the number of convulsions triggered by the administration of the vehicle, 10 mg·kg−1 AP14145 and 10 mg·kg−1 NS8593.

Article Snippet: NS8593 and NS 309 was supplied by NeuroSearch A/S, Ballerup, Denmark.

Techniques:

Journal: British Journal of Pharmacology

Article Title: A new negative allosteric modulator, AP14145, for the study of small conductance calcium‐activated potassium (K Ca 2) channels

doi: 10.1111/bph.14043

Figure Lengend Snippet: Calculated physicochemical properties and plasma protein binding of NS8593 and AP14145

Article Snippet: NS8593 and NS 309 was supplied by NeuroSearch A/S, Ballerup, Denmark.

Techniques: Protein Binding