Journal: Acta Pharmaceutica Sinica. B

Article Title: Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury

doi: 10.1016/j.apsb.2020.05.002

Figure Lengend Snippet: Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 compared with their controls. The numbers at the bottom of the bars indicate the number of repeats or mice in the group.

Article Snippet: After blocking by 5% milk, PVDF membranes were incubated with primary antibodies at 4 °C overnight such as rat anti-IL-4 antibody (1:500, Abcam, ab11524), mouse monoclonal anti-KCa3.1 antibody (1:250, Alomone, ALM-051), rabbit anti-Nav1.1 antibody (1:250, Alomone, ASC-001), rabbit GABA A R α 6 Polyclonal Antibody (1:200, Alomone, AGA-004), mouse anti- β -actin antibody (1:5000, Abcam) and mouse anti-GAPDH antibody (1:5000, Abcam).

Techniques: Expressing, Immunostaining, Staining, Marker, MANN-WHITNEY

Journal: Acta Pharmaceutica Sinica. B

Article Title: Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury

doi: 10.1016/j.apsb.2020.05.002

Figure Lengend Snippet: A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.

Article Snippet: After blocking by 5% milk, PVDF membranes were incubated with primary antibodies at 4 °C overnight such as rat anti-IL-4 antibody (1:500, Abcam, ab11524), mouse monoclonal anti-KCa3.1 antibody (1:250, Alomone, ALM-051), rabbit anti-Nav1.1 antibody (1:250, Alomone, ASC-001), rabbit GABA A R α 6 Polyclonal Antibody (1:200, Alomone, AGA-004), mouse anti- β -actin antibody (1:5000, Abcam) and mouse anti-GAPDH antibody (1:5000, Abcam).

Techniques: Binding Assay

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Coronary endothelial dysfunction prevented by small-conductance calcium-activated potassium channel activator in mice and patients with diabetes

doi: 10.1016/j.jtcvs.2020.01.078

Figure Lengend Snippet: Selective small-conductance calcium-activated potassium (SK) channels activator NS309 increased whole-cell K + currents in mouse heart endothelial cells ( MHECs ) of nondiabetics ( ND ) and diabetics ( DM ), which was blocked by simultaneous coapplication of the selective SK2/SK3 blocker apamin ( AP ) and SK4 blocker TRAM34 ( TR ). A, Representative current traces were obtained under control conditions in the presence of NS309 (10 −6 M) and AP (10 −7 M) plus TR (10 −5 M). B, Current–voltage (I-V) plot of the blocker-sensitive currents shown in A, demonstrating less current in DM cells compared with ND cells. C, Cumulative bar graph of NS309-activated currents at +100 mV showing that the amplitude of the SK currents in response to the SK activator NS309 was significantly reduced in DM MHECs compared with ND MHECs. n = 5/group. * P < .05. D, Relaxation response of mouse small coronary artery to NS309 (10 −6 M) at baseline (sham control) and after cardioplegic hypoxia/reoxygenation (CP-H/R). n = 5/group, # P < .05 vs ND; ** P < .001 vs control; @ P < .05 vs ND.

Article Snippet: The methods for whole-cell protein purification, Western blotting, and imaging quantification have been described previously., Membranes were incubated overnight at 4°C with primary antibodies against SK3 and SK4 (IK-1; Alomone Labs, Jerusalem, Israel).

Techniques: Control, Patch Clamp

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Coronary endothelial dysfunction prevented by small-conductance calcium-activated potassium channel activator in mice and patients with diabetes

doi: 10.1016/j.jtcvs.2020.01.078

Figure Lengend Snippet: A, Bar graphs showing recovery of the relaxation response of mouse small coronary arteries to adenosine 5′-diphosphate ( ADP ; 10 −4 M) after 60 minutes of cardioplegic (CP) hypoxia and reperfusion (CP-H/R) in sham controls, CP alone, CP + NS309 (10 −7 M), and CP + NS309 (10 −6 M) groups in diabetic ( DM ) and nondiabetic ( ND ) vessels. CP represents CP-H/R. B, Bar graphs showing recovery of the relaxation response of mouse small coronary arteries to substance P (10 −7 M) in sham controls, CP alone, CP + NS309 (10 −7 M), and CP + NS309 (10 −6 M) groups in DM and ND vessels. * P < .05 vs sham control (ND) or sham control (DM); # P < .05 vs sham control (ND); @ P < .05 vs CP (ND); U P < .05 vs CP (ND) or CP (DM); $ P < .05 vs CP + NS309 (ND). Data are mean ± SD. n = 6 to 8/group. C and D, Effects of small conductance calcium-activated potassium channels (SK) 3 blocker apamin ( Apa ) (10 −7 M) alone (C) and Apa + SK4 blocker TRAM34 (10 −5 M) (D) pretreatment on NS309-induced recovery of the relaxation response to ADP and substance P in mouse small coronary artery after 60 minutes of cardioplegic hypoxia and reoxygenation (CP) in the DM and ND groups; n = 5/group, data are mean ± SD, * P < .05 vs NS309 alone; # P < .05 vs NS309 + Apa. E, Effects of pretreatment with L-NAME on NS309-induced recovery of the relaxation response to substance P in mouse small coronary artery after 60 minutes of cardioplegic hypoxia and reperfusion in the DM and ND groups, n = 5/group, * P < .05 vs NS309 alone. F, Effects of pretreatment with indomethacin on NS309-induced recovery of relaxation response to substance P in mouse small coronary artery after 60 minutes of cardioplegic hypoxia and reperfusion in the DM and ND groups. n = 5/group. Data are mean ± SD.

Article Snippet: The methods for whole-cell protein purification, Western blotting, and imaging quantification have been described previously., Membranes were incubated overnight at 4°C with primary antibodies against SK3 and SK4 (IK-1; Alomone Labs, Jerusalem, Israel).

Techniques: Control

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Coronary endothelial dysfunction prevented by small-conductance calcium-activated potassium channel activator in mice and patients with diabetes

doi: 10.1016/j.jtcvs.2020.01.078

Figure Lengend Snippet: A, Subcellular protein expression of small conductance calcium-activated potassium channels (SK) 3 and SK4 in human coronary arterial endothelial cell ( HCAEC ) membrane and cytosolic lysates in the setting of normoxia or 60-minute hypoxia with 60 minutes of reoxygenation. Hypoxia represents hypoxia/reoxygenation in the diabetic ( DM ) and nondiabetic ( ND ) groups. B and C, Densitometric evaluation of immunoblot band intensity of SK3 and SK4. * P < .05 vs normoxia (ND); ** P < .001 vs normoxia (ND); @ P < .05 vs hypoxia (ND). Data are mean ± SD. n = 4 per group.

Article Snippet: The methods for whole-cell protein purification, Western blotting, and imaging quantification have been described previously., Membranes were incubated overnight at 4°C with primary antibodies against SK3 and SK4 (IK-1; Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Membrane, Western Blot

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Coronary endothelial dysfunction prevented by small-conductance calcium-activated potassium channel activator in mice and patients with diabetes

doi: 10.1016/j.jtcvs.2020.01.078

Figure Lengend Snippet: A and B, Densitometric evaluation of immunoblot band intensity of small conductance calcium-activated potassium channel ( SK ) 3 and SK4 in mouse heart endothelial cells ( MHECs ) (A) and human coronary artery endothelial cells ( HCAECs ) (B); C and D, densitometric evaluation of immunoblot band intensity of SK3 (C) and SK4 (D) in the MHECs; E and F, densitometric evaluation of immunoblot band intensity of SK3 (E) and SK4 (F) in the HCAECs, data are mean ± SD. n = 4/group. ND, Nondiabetic; DM , diabetic.

Article Snippet: The methods for whole-cell protein purification, Western blotting, and imaging quantification have been described previously., Membranes were incubated overnight at 4°C with primary antibodies against SK3 and SK4 (IK-1; Alomone Labs, Jerusalem, Israel).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Cardiomyocyte-expressed TGFβ signals to fibroblasts to program early heart maturation and adult myocyte identity

doi: 10.1101/2025.09.22.677845

Figure Lengend Snippet: a . Western blot showing time course of TGFβ1 or Latency associated peptide (LAP) (TGFβ) expression in the heart at embryonic day 16.5 (E16.5), postnatal day 1 (P1), P7, P21, and 12 weeks of age. Gapdh was used to normalize protein loading. b . Western blot for TGFβ1 and Cre from protein extracts of neonatal cardiomyocytes from Tgfb1/2/3 fl/fl-αMHC-Cre mice transduced with Ad-GFP or Ad-Cre 48 hrs prior in culture. c . mRNA expression of Tgfb1 , Tgfb2 , and Tgfb3 from neonatal cardiomyocytes transduced with Ad-GFP (adenovirus expressing green fluorescent protein) or Ad-Cre (adenovirus expressing Cre recombinase). n=2-5 per treatment group, *p<0.05. d . Schematic of the generation of Tgfb1/2/3 fl/fl-αMHC-Cre mice whereby Tgfb1 fl/fl , Tgfb2 fl/fl , and Tgfb3 fl/fl mice were crossed to the αMHC-Cre transgene. e . Kaplan-Meier curve depicting survival of Tgfb1/2/3 fl/fl and Tgfb1/2/3 fl/fl-αMHC-Cre mice over time in weeks. f . Heart-weight to body-weight (HW/BW) ratio of Tgfb1/2/3 fl/fl , Tgfb1/2/3 fl/fl-αMHC-Cre , and αMHC-Cre mice at 6 weeks of age. n=4-19 mice in each group, *p<0.05. g . Fractional shortening (FS%) at 6 weeks of age of the same groups of mice as in f. n=6-17 mice per group. *p<0.05. h . Left ventricular internal diameter in diastole (LVID;d) at 6 weeks of age in the same groups of mice as in f. n=6-17 mice per group. *p<0.05. i . Representative histological sections from hearts stained with Sirius Red to assess collagen deposition and wheat germ agglutinin-FITC (WGA, green) to assess cardiomyocyte cross sectional area (CSA) of Tgfb1/2/3 fl/fl and Tgfb1/2/3 fl/fl-αMHC-Cre mice at 6 weeks of age. Scale bar Sirius Red: 2 mm; Scale bar WGA: 100 µm. j . Quantification of average cardiomyocyte CSA from WGA staining as shown in i. A minimum of 250 myocytes were measured from each heart, across 3 hearts (points on the graph). *p<0.05. k . Hydroxyproline quantification of fibrosis from the left ventricle of hearts at 6 weeks of age as shown in j. n=8-9 mice per group. *p<0.05. l . Flow cytometry quantification of MEFSK4+ fibroblasts at 6 weeks of age in the hearts of mice shown in j. n=9-10 mice per group. *p<0.05. c,j,l. Data are presented as mean +/-SEM. Statistical analysis was performed using Student’s T-tests. f-h. Statistical analysis was performed using One Way ANOVAs with Tukey’s multiple comparisons test. k. In the case of non-normally distributed data, Mann Whitney tests were performed. Data are presented as mean +/-SEM.

Article Snippet: Fluorophore-conjugated antibodies against MEFSK4-allophycocyanin (APC) (Miltenyi Biotec, Waltham, MA, USA; 130-120-802;1:50), CD45-fluorescein isothiocyanate (FITC) (BD Biosciences, Franklin Lakes, NJ, USA; 563890;1:200), and CD31-brilliant violet 421 (BioLegend, San Diego, CA, USA; 102423;1:200) were added to samples and incubated on ice for 30 minutes.

Techniques: Western Blot, Expressing, Transduction, Staining, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cardiomyocyte-expressed TGFβ signals to fibroblasts to program early heart maturation and adult myocyte identity

doi: 10.1101/2025.09.22.677845

Figure Lengend Snippet: a . Images of hearts at P1 and P7 from the two genotypes of mice demonstrating early dysmorphia. Scale bar: 1 mm (equal aspect ratio in both pictures). b . Heart-weight to body weight ratio (HW/BW) at P7 in the three genotypes of mice shown. n=8-14 mice per group. *p<0.05. c . Hydroxyproline quantification of fibrosis from left ventricles of mice at P7 in the three genotypes of mice shown. n=4-6 mice per group. d . Flow cytometric quantification of MEFSK4+ fibroblasts from left ventricles of mice at P7 in the two genotypes of mice shown. n=8 mice per group. e . Atomic force microscopy quantification of cardiac stiffness at P7 in hearts of Tgfb1/2/3 fl/fl and Tgfb1/2/3 fl/fl-αMHC-Cre mice. A total of 64 force curves were generated from histological sections in minimally 10 different areas of the LV free wall (minimally 640 force curves analyzed/heart). Each datum point depicted represents the average of the force curves generated for a given heart. n=5 mice per group. *p<0.05. f . Western blot assessment of extracellular matrix proteins in the hearts of Tgfb1/2/3 fl/fl and Tgfb1/2/3 fl/fl-αMHC-Cre mice at P7. GAPDH was used as a processing and loading control. g . Representative immunostaining of cardiac histological sections for WT1 (green) merged with laminin (red) to assess epicardial derived cells migrating into the myocardium at E18.5 for the two genotypes shown. Scale bars: 100 µm. h . mRNA expression of genes involved in epithelial-mesenchymal transition (EMT), smooth muscle cell (SMC) maturation, and extracellular matrix (ECM) expression in E14.5 hearts of Tgfb1/2/3 fl/fl and Tgfb1/2/3 fl/fl-αMHC-Cre mice. Genes were normalized to 18S ribosomal RNA expression. n=4-6 mice per group. b,c. Data presented as mean +/-SEM. Kruskal Wallis tests were used with Dunn’s multiple comparisons test for statistical analysis. d,e,h. Data presented as mean +/-SEM. Student’s T-test used for statistical analysis.

Article Snippet: Fluorophore-conjugated antibodies against MEFSK4-allophycocyanin (APC) (Miltenyi Biotec, Waltham, MA, USA; 130-120-802;1:50), CD45-fluorescein isothiocyanate (FITC) (BD Biosciences, Franklin Lakes, NJ, USA; 563890;1:200), and CD31-brilliant violet 421 (BioLegend, San Diego, CA, USA; 102423;1:200) were added to samples and incubated on ice for 30 minutes.

Techniques: Microscopy, Generated, Western Blot, Control, Immunostaining, Derivative Assay, Expressing, RNA Expression

Journal: Veterinary Research

Article Title: Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol

doi: 10.1186/s13567-016-0309-1

Figure Lengend Snippet: TLR2 ligands enhanced barrier function in IPEC-J2 cells. IPEC-J2 cells were treated with synthetic TLR2 ligands, Pam3CSK4 (PCSK), B. subtilis -derived PGN (PGN-BS) or B. subtilis -derived LTA (LTA-BS) at 0, 0.1, 1, or 10 μg/mL. A TEER was examined at 24 h using epithelial voltohm meter. Data are presented as mean ± SD ( n = 4). * P < 0.05; ** P < 0.01, determined by one-way ANOVA with Tukey’s posttest. B The monolayer of IPEC-J2 cells was lysed and protein extracts were analyzed for claudin-3, occludin, and ZO-1 by using Western blot assay. C Lysates were produced from membrane and cytosolic portion of the cells and the expression of claudin-3 and occludin at 24 h was examined by Western blot assay. β-actin was used as an internal control ( n = 3). The representative figure from three similar results is shown. Cyt; cytosol, Mem; membrane.

Article Snippet: IPEC-J2 cell monolayer was treated with 2 μg/mL of DON (Sigma, Missouri, USA) for 24, 48 or 72 h. To evaluate the effect of TLR2 agonists on the barrier function, IPEC-J2 cells were pretreated with 10 μg/mL of LTA from B. subtilis (LTA-BS; Invivogen, San Diego, USA), PGN from B. subtilis (PGN-BS; Invivogen), Pam3CSK4 (Pam3Cys-SKKKK; Invivogen) or complete medium as a control for 24 h before DON treatment.

Techniques: Derivative Assay, Western Blot, Produced, Membrane, Expressing

Journal: bioRxiv

Article Title: Potassium channels mediate the inhibitory effect of exosomes on anti-tumor immunity in head and neck cancer

doi: 10.1101/2025.07.25.666838

Figure Lengend Snippet: (A) (Left) Representative KCa3.1 currents recorded in whole-cell voltage-clamp configuration in activated CD8 + T cells from a healthy donor in the absence (Control) or presence of HNSCC exosomes (EXO; 1.04 X 10 9 particles/ ml). (Right) Violin plots of KCa3.1 and Kv1.3 conductance (normalized to cell capacitance) in activated CD8 + T cells from five healthy donors in the absence (Control) or presence of HNSCC exosomes (EXO) from HNC208, HNC285, and HNC365 (n= 21 cells for control and 23 cells for EXO; some donors’ T cells were treated with exosomes from multiple HNSCC patients). Significance was determined using the Mann-Whitney rank sum test. (B) Fold change in mRNA abundance of CALM1 , CALM2, and CALM3 in PBMCs from healthy donors (n=7) treated with HNC208, HNC285, and HNC365 exosomes (EXO; 1.04 X 10 9 particles/ ml) was determined by RT-qPCR. The PBMCs from some donors in the EXO group were each treated with exosomes from two different HNSCC patients. Each sample was run in triplicate. 18S rRNA was used as the housekeeping gene. Data were normalized to untreated PBMCs (Control). Bars represent means ± SD, and the symbols represent individual treatment conditions. Significance was determined using an unpaired t-test ( CALM2 ) and Mann-Whitney rank sum test ( CALM1 and CALM3 ). (C) Representative flow cytometry staggered histograms showing CaM expression in CD8 + T cells from one healthy donor in the absence (Control) or presence of HNC 208, HNC 285, and HNC 365 exosomes (EXO; 1.04 X 10 9 particles/ ml). The bar graph on the right shows the geometric mean fluorescence intensity (gMFI) for CaM expression in control and EXO-treated cells from four healthy donors. Bars represent mean ± SD; symbols represent individual treatment conditions (some donors were treated with exosomes from multiple HNSCC patients). Significance was determined by Mann-Whitney rank sum test. (D) Representative Ca 2+ response (shown as a ratio of Indo-1 fluorescence at 400 and 480 nm) recorded in activated healthy donor CD8 + T cells in the absence (Control) or presence of HNC208, HNC285, and HNC365 exosomes (EXO; 1.04 X 10 9 particles/ ml). Cells were loaded with Indo-1; and analyzed by flow cytometry, thapsigargin (arrow) was added in 0 mM Ca 2+ to induce intracellular Ca 2+ store depletion, followed by 2 mM Ca 2+ , which yielded a rapid Ca 2+ influx (see Materials and Methods). Right panel shows the average peak Ca²⁺ fold change in CD8 + T cells from n = 5 donors. Bars represent mean ± SD; symbols represent individual treatment conditions (some donors were treated with exosomes from multiple HNSCC patients). Statistical significance was determined by unpaired t-test. (E) IFN-γ levels in healthy donor CD8 + T cells (n =3 healthy donors) treated with HNSCC exosomes (0.13 × 10 9 particles/ml), isolated from supernatants of HNC208, HNC285, and HNC365 cells, and activated with plate-bound anti-CD3 and anti-CD28 antibodies for 72 h in the absence or presence of 1 μM NS309 (KCa3.1 activator). Each donor was exposed to HNC208, HNC285, and HNC365 exosomes, and the samples were run in duplicate on the same ELISA plate. Activated CD8 + T cells that were not exposed to exosomes were used as controls. For each experiment, IFN-γ levels in control samples were normalized to 1 (dotted line), and all other values were expressed relative to this baseline. Symbols represent individual treatment conditions; bars indicate mean ± SD. Significance was determined using a paired t-test.

Article Snippet: The cells were then resuspended in 100 μl PBS, and stained with Zombie UV Live/Dead dye (BioLegend) as per the manufacturer’s instructions, washed with staining buffer (BioLegend), fixed in 4% paraformaldehyde for 30 minutes and stained overnight at 4°C with guinea pig anti-Kv1.3 and mouse anti-KCa3.1 (ATTO-488) primary antibodies (Alomone Labs) in the dark.

Techniques: Control, Exosomes, MANN-WHITNEY, Quantitative RT-PCR, Flow Cytometry, Expressing, Fluorescence, Isolation, Enzyme-linked Immunosorbent Assay, FACS, Patch Clamp

Journal: bioRxiv

Article Title: Potassium channels mediate the inhibitory effect of exosomes on anti-tumor immunity in head and neck cancer

doi: 10.1101/2025.07.25.666838

Figure Lengend Snippet: (A) Fold change in mRNA abundance of KCa3.1 (KCNN4 ), Kv1.3 ( KCNA3 ), Orai ( ORAI1 ) and Stim1 ( STIM1 ) in PBMCs from healthy donors (n=7) treated with HNC208, HNC285 and HNC365 exosomes (EXO, 1.04 X 10 9 particles/ ml) was determined by RT-qPCR. PBMCs from some donors in the EXO group were each treated with exosomes from two different HNSCC patients. Each sample was run in triplicate. 18S rRNA was used as the housekeeping gene. Data were normalized to untreated PBMCs (Control). Bars represent means ± SD, and symbols represent individual treatment conditions. Significance was determined by unpaired t-test ( ORAI1 and STIM1 ) and Mann-Whitney rank sum test ( KCNN4 and KCNA3 ). (B-E) Mean fluorescence intensity (MFI) of KCa3.1, Kv1.3, and CD69 in CD3 + (B), CD4 + (C), CD8 + (D), and CD56 dim CD16 + NK cells (E) from four healthy donors treated with HNSCC exosomes from HNC208, HNC285, and HNC365 (same protocol as in A, some donors were treated with exosomes from multiple HNSCC patients). Untreated PBMC were used as controls. Significance was determined by unpaired t-test and Mann-Whitney rank sum test (CD3 + CD69, CD4 + KCa3.1, CD4 + CD69, NK + Kv1.3, NK + CD69). Bars represent means ± SD, symbols represent individual treatment conditions.

Article Snippet: The cells were then resuspended in 100 μl PBS, and stained with Zombie UV Live/Dead dye (BioLegend) as per the manufacturer’s instructions, washed with staining buffer (BioLegend), fixed in 4% paraformaldehyde for 30 minutes and stained overnight at 4°C with guinea pig anti-Kv1.3 and mouse anti-KCa3.1 (ATTO-488) primary antibodies (Alomone Labs) in the dark.

Techniques: Exosomes, Quantitative RT-PCR, Control, MANN-WHITNEY, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: αPD-1 treatment increases K + channel activity in HNSCC T cells. (A) Representative current traces of KCa3.1 and Kv1.3 channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs cells from a HNSCC patient in absence or presence of αPD-1 (10 μg/ml, for 6 h). Data are normalized to maximum current at +50 mV recorded using a ramp pulse protocol from −120 mV to +50 mV for 200 ms every 15 s. The holding potential used was −70 mV. (B,C) KCa3.1 (B) and Kv1.3 (C) conductance (G) measured in the absence or presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 68 cells without pembrolizumab and n = 55 cells with pembrolizumab from 14 patients). (D) Representative current traces of divalent free current (DVF) through CRAC channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs from a HNSCC patient. Data were recorded using a ramp pulse protocol from −100 to +100 mV with at holding potential of +30 mV every 1.5 s. Cells were perfused with 0 mM Ca 2+ solution (1 min) followed by 20 mM Ca 2+ (1 min) and DVF solutions (2 min, see methods) to amplify currents during recordings. (E) Peak DVF current values measured in absence and presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 34 cells without αPD-1 and n = 31 cells with αPD-1 from 8 patients). The values in panels (B,C) and (E) are represented as box plots: the horizontal line indicates the median; the lower box is the 25 th percentile; the upper box is the 75 th percentile; and the whiskers represent the 10 th and 90 th percentiles. (F) Ion channel expression (KCa3.1, Kv1.3, Orai1 and STIM1) in HNSCC patient T cells after treatment with αPD-1 (10 μg/ml for 6 h). Effect of αPD-1 treatment is shown as ratio of mean fluorescence intensity (MFI, fold change) values of treatment versus control group. Data are represented as scatter plot where each symbol represents an individual patient ( n = 4–5). Horizontal line represents mean values for each group. Data in panels (B,C,E) were analyzed by Mann-Whitney rank sum test.

Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

Techniques: Activity Assay, Incubation, Expressing, Fluorescence, Control, MANN-WHITNEY, Patch Clamp

Journal: Frontiers in Pharmacology

Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: αPD-L1 treatment increases K + channel activity in HNSCC patients. (A) KCa3.1 and (B) Kv1.3 conductance values (G) measured with and without the αPD-L1 antibody, atezolizumab (1 and 10 μg/ml for 6 h) in activated CD8 + PBTs of HNSCC patients. (C) KCa3.1 and (D) Kv1.3 G measured in presence of PD-L1 and αPD-1 antibody pembrolizumab in activated CD8 + PBTs of HDs. Activated cells were treated with plate-bound PD-L1 (PD-L1-Fc 10 μg/ml) +/- αPD-1 (untreated cells were used as a control) and activated for 72 h using PMA/Ionomycin. αPD-1 was added to treatment group for 6 h. Data in the lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal lines. The lower and upper error bars represents 10 th and 90 th percentile respectively, n = 8–23 cells from 3 HNSCC patients, n = 30 cells from 6 HDs (control and PD-L1) and n = 15 cells from 3 HDs (PD-L1 + αPD-1). Five cells were recorded for each individual donor. Data in (A,C,D) were analyzed by ANOVA on ranks test ( p < 0.001) followed by Dunn’s post hoc analysis. Data in (B) were analyzed by One way ANOVA ( p < 0.001) followed by Holm-Sidak test.

Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

Techniques: Activity Assay, Control, Analysis

Journal: Frontiers in Pharmacology

Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: Short-time treatment with PD-L1 decreases KCa3.1 activity in a calmodulin-independent manner. (A) Flow cytometry histogram and geometric mean fluorescence intensity (gMFI) values (B) for CaM expression in activated CD8 + PBTs from HD donors ( n = 3) in the absence and presence of PD-L1. (C) Representative recordings of KCa3.1 currents in activated CD8 + PBTs from HDs showing the effect of PD-L1 (PD-L1-Fc, 10 μg/ml) and CaM (50 µM). (D) Average normalized KCa3.1 conductance ( G , nS) measured in the absence and presence of PD-L1, with and without CaM. All conductance values are normalized to average conductance value obtained from control recordings. Cells were pre-incubated with plate-bound PD-L1 (PD-L1-Fc,10 μg/ml, for 72 h) activated using anti-CD3/CD28 antibodies and treated with or without CaM (50 µM), that was delivered intracellularly via patch pipette during recordings ( n = 15–18 cells per group from 3 HDs). The values in panel (B) are represented as bar graphs. Each symbol represent an individual HD. The values are represented as mean ± SEM. The values in panel (D) are represented as box and whisker plots. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. Data in panel (B) were analyzed by t -test and data in panel (D) were analyzed by ANOVA on ranks ( p = 0.008) with Dunn’s post hoc analysis.

Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Expressing, Control, Incubation, Transferring, Whisker Assay, Analysis, FACS, Patch Clamp

Journal: Frontiers in Pharmacology

Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.

Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

Techniques: Inhibition, Patch Clamp, Transferring, Control, Fluorescence, Flow Cytometry, Analysis

Journal: Heliyon

Article Title: SK4 calcium-activated potassium channels activated by sympathetic nerves enhances atrial fibrillation vulnerability in a canine model of acute stroke

doi: 10.1016/j.heliyon.2020.e03928

Figure Lengend Snippet: Effect of stellate ganglion ablation and TRAM-34 on AF induction. The ∑WOV was calculated in the three groups. The SK4 inhibitor TRAM-34 completely inhibited the inducibility of AF in AS dogs (∗P < 0.05 vs. the control group and LSGA group).

Article Snippet: The primary antibody SK4 (bs-6675R-HRP, Bioss, Inc., China, 1:200) and c-Fos (ab209794, Abcam, Inc., UK; 1:100) were added to the sections overnight at 4 °C.

Techniques:

Journal: Heliyon

Article Title: SK4 calcium-activated potassium channels activated by sympathetic nerves enhances atrial fibrillation vulnerability in a canine model of acute stroke

doi: 10.1016/j.heliyon.2020.e03928

Figure Lengend Snippet: The expression of SK4 in the RAAW and LAAW in the three groups. (A–F) SK4 expression in the control group, AS group and LSGA group detected by immunohistochemistry (black arrow). (original magnification: ×400). (G) Quantitative analysis of SK4 expression. (∗P < 0.05 vs. RA in the control group and LSGA group, ＃P < 0.05 vs. RA in the AS group, † P＜0.05 compared with LA in the control group and LSGA group, § P＜0.05 compared with RA in the control group and LA in the LSGA group). (H and I) The levels of SK4 protein detected by WB analysis. (∗P < 0.05 vs. the control group and LSGA group, ＃P < 0.05 vs. the control group). See supplementary data for uncropped blot.

Article Snippet: The primary antibody SK4 (bs-6675R-HRP, Bioss, Inc., China, 1:200) and c-Fos (ab209794, Abcam, Inc., UK; 1:100) were added to the sections overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry