anti kcnn4  (Alomone Labs)

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Intermediate Conductance Calcium‐Activated Potassium Channel Activation in Stem Cell Antigen‐1 Positive Cells Contributed to Vascular Restenosis

doi: 10.1161/JAHA.125.045943

Figure Lengend Snippet: A , Effect on migration. ATP increased Sca‐1 + cell migration, which was significantly inhibited by the IK Ca channel blocker TRAM34 ( a ). MCP‐1 also enhanced migration, and TRAM34 similarly suppressed this increase ( b ). (Statistical significance was confirmed by HC3‐robust interaction model with 6 biological replicates.) B , The ATP‐enhanced migration of Sca‐1 + cells was significantly abolished by IK Ca channel inhibition with shKCNN4 ( P =0.0360, HC3‐robust interaction model, n=5). C , The proliferation of Sca‐1 + cells induced by ATP ( a , b ) and PDGF‐BB ( c ) was significantly suppressed by inhibition of IK Ca channels with TRAM34 or shKCNN4 ( P values are indicated on the graph; HC3‐robust interaction model, n=5). ATP indicates adenosine triphosphate; CON, control; HC3, heteroskedasticity‐consistent robust SE; IK Ca , intermediate conductance calcium‐activated potassium channel; MCP‐1, monocyte chemoattractant protein‐1; PDGF‐BB, platelet‐derived growth factor‐BB; Sca‐1 + , stem cell antigen‐1 positive; shKCNN4, a short hairpin RNA construct specifically designed to target and silence the expression of the KCNN4 gene; and TRAM34, 1‐ [(2‐chlorophenyl) diphenylmethyl]‐1h‐pyrazole.

Article Snippet: They were then blocked with BSA and incubated overnight with an anti‐ KCNN4 receptor (extracellular) antibody (1:200, Alomone, Jerusalem, Israel, #APR‐102).

Techniques: Migration, Inhibition, Control, Derivative Assay, shRNA, Construct, Expressing

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: Predesigned TaqMan Gene Expression Assay primers (Applied Biosystems, Thermo Fisher Scientific) were used to detect the expression of KCNN4 (assay ID: Hs01069779_m1) and 18S rRNA (assay ID: Hs99999901_s1).

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: HNSCC patients have reduced levels of functional KCa3.1 channels in NK cells compared to healthy donors. ( A ) Average KCa3.1 activity (reported as conductance) and ( B ) average cell capacitance recorded in resting and activated NK cells from HNSCC patients (17 resting and 29 activated cells from 8 HNSCC patients). ( C , D ) Comparison of KCa3.1 activity and cell capacitance between (C) healthy donors (HD) and (D) HNSCC patients in activated NK cells (14 cells from 4 HDs and 29 cells from 8 HNSCC patients). The data for HDs are the same data reported in Fig. B. ( E ) Single-cell trajectories of activated NK cells from a representative healthy donor (left) and HNSCC patient (right) migrating towards a chemokine (CXCL10) gradient. 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. ( F ) Average Y-COM of activated NK cells migrating towards a CXCL10 gradient in HD ( n = 5) and HNSCC ( n = 5) individuals. Data were analyzed by either unpaired Student’s t-test (A , B , D , F) or Mann-Whitney test (C). Bars represent mean ± SD. Each symbol represents either an individual cell (A-D) , or an individual donor for (F) .

Article Snippet: Predesigned TaqMan Gene Expression Assay primers (Applied Biosystems, Thermo Fisher Scientific) were used to detect the expression of KCNN4 (assay ID: Hs01069779_m1) and 18S rRNA (assay ID: Hs99999901_s1).

Techniques: Functional Assay, Activity Assay, Comparison, MANN-WHITNEY

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: Predesigned TaqMan Gene Expression Assay primers (Applied Biosystems, Thermo Fisher Scientific) were used to detect the expression of KCNN4 (assay ID: Hs01069779_m1) and 18S rRNA (assay ID: Hs99999901_s1).

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: Predesigned TaqMan Gene Expression Assay primers (Applied Biosystems, Thermo Fisher Scientific) were used to detect the expression of KCNN4 (assay ID: Hs01069779_m1) and 18S rRNA (assay ID: Hs99999901_s1).

Techniques: Activation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Proliferation 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 activation reduces tumor burden and increases the intratumoral abundance of cytotoxic NK cells in a HNSCC mouse model. ( A ) Schematic of HNSCC humanized mouse model generation of NSG mice injected with Cal27 cells (a human HNSCC cell line) on day 0, followed by engraftment of human healthy donor PBMCs +/- SKA-31 (30 mg/kg) injections on day 21. The tumor tissue size was measured between days 21–35 and on day 35, the tumors were harvested for immunohistochemical (IHC) staining. ( B ) Effect of SKA-31 treatment on tumor burden (size measured in mm 3 ) (4 mice/group; * P < 0.05 by unpaired t-test). Day 0 corresponds to day 21 in panel A, i.e., the day PBMCs were injected. ( C ) Immunohistochemistry of CD161 (dark brown signal; NK cells) and granzyme B (purple signal) expression in tumors harvested from HNSCC humanized mice. Representative IHC images obtained from a region of interest (ROI) within the tumor area of xenografts, highlighting CD161 and granzyme B staining in the presence or absence of SKA-31 are shown here. Scale bar = 50 μm. ( D ) Intra-tumoral abundance of NK cells in vehicle (-SKA-31) and SKA-31-treated mice, determined as number of cells divided by the area of the ROIs within the intratumoral region of the tissues. ( E ) Granzyme B-positive NK cell density +/- SKA-31 normalized for the area of ROIs. ( F ) Immunohistochemistry of CD161 (dark brown signal; NK cells) and granzyme B (purple signal) expression in tumors harvested from HNSCC humanized mice. Representative IHC images obtained from a region of interest (ROI) from the periphery of tumor biopsies, highlighting CD161 and granzyme B staining in the presence or absence of SKA-31 are shown here. (G) Abundance of NK cells in the tumor periphery, in vehicle (-SKA-31) and SKA-31-treated mice, determined as number of cells divided by the area of the ROIs within the periphery region of the tissues. ( H ) Granzyme B-positive NK cells, in the periphery of the tumor tissues, in SKA-31-treated and untreated (vehicle, -SKA-31) mice. For each biopsy, 10 fields (ROIs) were imaged, and the quantification was analyzed using an unpaired Student’s t-test. Bars represent mean ± SD, and each symbol represents an individual measurement from the analyzed fields from 4 mice/group.

Article Snippet: Predesigned TaqMan Gene Expression Assay primers (Applied Biosystems, Thermo Fisher Scientific) were used to detect the expression of KCNN4 (assay ID: Hs01069779_m1) and 18S rRNA (assay ID: Hs99999901_s1).

Techniques: Activation Assay, Injection, Immunohistochemical staining, Immunohistochemistry, Expressing, Staining