Journal: American Journal of Physiology - Cell Physiology

Article Title: Dextran sulfate sodium-induced chronic colitis attenuates Ca 2+ -activated Cl − secretion in murine colon by downregulating TMEM16A

doi: 10.1152/ajpcell.00328.2017

Figure Lengend Snippet: Custom qPCR primers for determination of transcript abundance

Article Snippet: PVDF membranes were then incubated in primary antibody: TMEM16A 1:500, CFTR 1:200, bestrophin-2 (BEST2) 1:500, muscarinic 3 (M 3 ) receptor 1:200 (Alomone Laboratories), inositol (1,4,5)-trisphosphate (IP 3 ) receptor 1:500, β-actin 1:1,000 (Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Dextran sulfate sodium-induced chronic colitis attenuates Ca 2+ -activated Cl − secretion in murine colon by downregulating TMEM16A

doi: 10.1152/ajpcell.00328.2017

Figure Lengend Snippet: Effect of dextran sulfate sodium (DSS)-colitis on transcript abundance of Cl− channels in murine colon. Transcript abundance was determined for transmembrane protein 16A (Tmem16a; A), bestrophin-2 (Best2; B), and Cftr (C) in control (open bars) or DSS-colitis mice (closed bars). Bar graphs of summarized data represent means ± SE of 5 different animals normalized to an endogenous control, Actb. *P < 0.001, compared with their respective control.

Article Snippet: PVDF membranes were then incubated in primary antibody: TMEM16A 1:500, CFTR 1:200, bestrophin-2 (BEST2) 1:500, muscarinic 3 (M 3 ) receptor 1:200 (Alomone Laboratories), inositol (1,4,5)-trisphosphate (IP 3 ) receptor 1:500, β-actin 1:1,000 (Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Dextran sulfate sodium-induced chronic colitis attenuates Ca 2+ -activated Cl − secretion in murine colon by downregulating TMEM16A

doi: 10.1152/ajpcell.00328.2017

Figure Lengend Snippet: Effect of dextran sulfate sodium (DSS)-colitis on Cl− channel protein expression in murine colon. A: representative images of protein expression of the different characterized Cl− channels from epithelial lysates. BEST2, bestrophin-2. B: transmembrane protein 16A (TMEM16A) protein expression normalized to β-actin for quantification of control and DSS-colitis groups. TMEM16A expression (closed bar) is significantly decreased in DSS-colitis mice as compared with control. C and D: summarized data of Western blot quantification of BEST2 and CFTR normalized to β-actin in control and DSS-colitis mice. Neither protein was significantly altered from the control cohort. Bar graphs of summarized data represent means ± SE of 5 different animals from each respective group. *P < 0.05, compared with their respective control.

Article Snippet: PVDF membranes were then incubated in primary antibody: TMEM16A 1:500, CFTR 1:200, bestrophin-2 (BEST2) 1:500, muscarinic 3 (M 3 ) receptor 1:200 (Alomone Laboratories), inositol (1,4,5)-trisphosphate (IP 3 ) receptor 1:500, β-actin 1:1,000 (Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Dextran sulfate sodium-induced chronic colitis attenuates Ca 2+ -activated Cl − secretion in murine colon by downregulating TMEM16A

doi: 10.1152/ajpcell.00328.2017

Figure Lengend Snippet: Immunohistochemistry of transmembrane protein 16A (TMEM16A) in colonic tissue sections. A: immunohistochemistry of TMEM16A in control tissue demonstrates pronounced expression and localization of the protein to surface epithelium and upper crypts (top, left). B. DSS-colitis tissue stained for TMEM16A exhibits less staining in epithelial cells and a more diffuse signal in the lamina propria (bottom, left). C and D: both cohorts of tissue were run in parallel with a control peptide specific for the anti-TMEM16A antibody. Both images demonstrate a lack of staining when coincubated with control peptide. Images were captured at ×20 magnification on the AxioImager (Carl Zeiss, Germany).

Article Snippet: PVDF membranes were then incubated in primary antibody: TMEM16A 1:500, CFTR 1:200, bestrophin-2 (BEST2) 1:500, muscarinic 3 (M 3 ) receptor 1:200 (Alomone Laboratories), inositol (1,4,5)-trisphosphate (IP 3 ) receptor 1:500, β-actin 1:1,000 (Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques: Immunohistochemistry, Expressing, Staining

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 1. ANO1 is upregulated in metastatic tumors and correlated with poor prognosis in ESCC. A, Flow chart of the strategy for the screening of cancer metastasis drivers. B, Heatmap demonstrating the differentially expressed genes between metastatic tissues and primary ESCC tumors. C, Venn diagram was used to overlap the genes upregulated in ESCC lymph node metastasis tissues and highly metastatic ESCC cell subline. D and E, The expression of ANO1 was examined in three cases of primary ESCC tumors and paired lymph node metastasis tissues by qRT-PCR (D) and Western blot analysis (E). F, Representative immunohistochemical images and quantitative analysis of ANO1 staining in 100 ESCC tissues and 75 matched normal tissues. G, Survival analysis of 100 patients with ESCC stratified by the ANO1 level. H, Representative images and quantitative analysis of ANO1 staining in 40 ESCC tissues and the matched metastatic samples. I, The expression level of ANO1 in the cohort of esophageal carcinoma (ESCA) in the TCGA database. J, Analysis of the ANO1 expression in esophageal cancer patients with different nodal metastasis status on UALCAN website. K, Analysis of the ANO1 level in patients with HNSC, KIRC, PCPG, and STAD in TCGA database. Bars, SD. , P < 0.05; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 2. ANO1 promotes cancer metastasis in vitro and in vivo. A and B, The invasive ability of ANO1-overexpressing (A) or ANO1-knockdown (B) ESCC cells was examined by Boyden chamber invasion assay. C and D, The protein expression of b-catenin, E-cadherin, and snail was compared in ANO1-overexpressing (C) or ANO1-knockdown (D) ESCC cells by Western blot analysis. E, Representative images of swollen inguinal lymph nodes and primary tumors in mice receiving subcutaneous footpad injection of ANO1-overexpressing ESCC cells or control cells (6 mice per group). F, Hematoxylin and eosin staining of invaded tumor in lymph node metastasis model. G, Mice were intravenously injected with ANO1-overexpressing or control cells and the lung colonization was detected using bioluminescence imaging (6 mice per group). H, Hematoxylin and eosin staining of lung sections in lung colonization model. I–L, The effect of ANO1 knockdown on the metastasis of ESCC cells to the lymph nodes and lungs. Bars, SD. , P < 0.01; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: In Vitro, In Vivo, Knockdown, Invasion Assay, Expressing, Western Blot, Injection, Control, Staining, Imaging

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 3. ANO1 increases intracellular cholesterol level via inactivation of LXR pathway. A, Volcano plot of differentially expressed genes (fold change > 2; P < 0.05) in ANO1-overexpressing ESCC cells by RNA-seq. B, Gene ontology analysis indicated the alternation of LXR signaling in ANO1-overexpressing cells. C and D, The mRNA and protein levels of ABCA1 and ABCG1 in ANO1-overexpressing ESCC cells were examined by qRT-PCR and Western blot analysis. E and F, Effect of ANO1 knockdown on ABCA1 and ABCG1 expression in ESCC cells. G and H, Intracellular cholesterol level in ESCC cells with manipulation of ANO1 expression. I, Intracellular cholesterol level of ANO1-overexpressing ESCC cells in presence or absence of GW3965 (5 mmol/L). J, Relative mRNA levels of ABCA1 and ABCG1 in ANO1-overexpressing ESCC cells in presence or absence of GW3965 (5 mmol/L). K, The invasive abilities of ANO1-overexpressing ESCC cells and control cells were determined in the presence or absence of GW3965 (5 mmol/L) or M-b-CD (2.5 mmol/L). L and M, Comparison of the lung colonization of ANO1-overexpressing ESCC cells and control cells in the mice with or without GW3965 (10 mg/kg) or M-b-CD (64 mg/kg) treatment. Bars, SD. , P < 0.01; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Knockdown, Expressing, Control, Comparison

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 4. ANO1 interacts with JUN to inhibit CYP27A1 transcription and to repress cholesterol hydroxylation. A and B, qRT-PCR and Western blot analysis of the expression of CYP27A1 in ANO1-overexpressing ESCC and control cells. C and D, Effect of ANO1 knockdown on CYP27A1 expression in ESCC cells. E, The luciferase activity of CYP27A1 promoter was determined in the ESCC cells with overexpression of ANO1. (Continued on the following page.)

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control, Knockdown, Luciferase, Activity Assay, Over Expression

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 5. ANO1-expressing ESCC cells induce IL1b secretion and activate fibroblasts. A, Diagram showing the coculture systemof ESCC cells and fibroblasts. B, The migration of fibroblasts attracted by the CM from ANO1-overexpressing ESCC cells and control cells was determined by Boyden chamber assay. C, The expression of aSMA, FAP in the fibroblasts treated with indicated CM was examined by Western blot analysis. D, Heatmap of the differentially expressed proteins in the CM of ANO1- overexpressing ESCC cells detected by cytokine array assay. E and F, The expression and secretion levels of IL1b were determined by Western blot analysis (E) and ELISA (F) in ANO1-overexpressing ESCC cells and control cells. G and H, Knockdown of ANO1 in ESCC cells decreased IL1b expression and secretion. I and J, Effect of ANO1 manipulation on IL1b mRNA level in ESCC cells. K and L, The mRNA and protein expression of IL1b was determined in ANO1-overexpressing cells with or without GW3965 (5 mmol/L) treatment. M, Further knockdown of IL1b in ESCC cells or the addition of IL1b neutralizing antibody reversed the migration of fibroblasts induced by ANO1-overexpressing ESCC cells. N, Fibroblast activation markers aSMA and FAP were detected by Western blot analysis in the fibroblasts treated with the indicated CM. Bars, SD. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Expressing, Migration, Control, Boyden Chamber Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Activation Assay

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 6. IL1b-activated fibroblasts induces CCL1 secretion to exert positive feedback on ESCC cancer cell invasion. A, Flow chart showing the collection of CM from activated fibroblasts to attract the invasion of ESCC cells. B, Invasion of KYSE150 and EC9706 cells exposed to the different fibroblast-derived CM. C, Radar map illustrating the top 30 upregulated cytokines in the CM of fibroblasts treated with supernatant of ANO1-overexpresing ESCC cell by cytokine antibody array. D, After transfection with the siRNAs targeting the top 10 upregulated cytokines, respectively, the fibroblasts were treated with rIL1b (20 ng/mL) and the CM were collected to attract the invasion of KYSE150 cells. E, The secretion of CCL1 from the fibroblasts exposed to rIL1b (20 ng/mL) for 48 hours was determined by ELISA assay. F, Western blot was used to detect p-p65 and p65 expressionin the fibroblasts stimulated with rIL1b (20 ng/mL) in presence or absence of CAPE (10 mmol/L). G andH, qRT-PCR and ELISA analyses of CCL1 expression and secretion in the fibroblasts exposed to rIL1b (20 ng/mL) with or without addition of CAPE (10 mmol/L). I, Invasion of KYSE150 and EC9706 cells exposed to the different fibroblast-derived CM in presence or absence of CAPE. Bars, SD. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Derivative Assay, Ab Array, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Expressing

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 7. Lead compound K786–4469 targets ANO1 to suppress tumor metastasis. A, Schematic diagram of screening strategies for the ANO1-targeting small molecule compounds. B, The inhibitory effects of 24 candidate compounds on ESCC cell invasion were compared using Boyden chamber assay. C, The expression of ANO1, CYP27A1, ABCA1, and ABCG1 in ESCC cells treated with increasing concentrations of K786–4469 (up to 10 mmol/L) was detected by Western blot. D, Effect of K786– 4469 on mRNA levels of ABCA1, ABCG1, and CYP27A1. E, The intracellular cholesterol level was determined in K786–4469-treated ESCC cells. F, K786–4469 repressed ESCC cell invasion in a dose-dependent manner. G, Mice were intravenously injected with KYSE150-Luc-LM3 cells and treated with K786–4469 or DMSO; lung colonization was detected using bioluminescence imaging. H, Hematoxylin and eosin staining of lung sections as indicated. I, The structure of the ANO1 protein complexed with K786–4469. J, Wild-type or different mutant ANO1 was re-overexpressed in ANO1-knockdown ESCC cells, and suppressive effects of K786–4469 were compared by using Boyden chamber assay. K, Lung colonization in the mice injected with the indicated cell lines and treated with K786–4469 or DMSO was detected using bioluminescence imaging. Bars, SD. n.s., nonsignificant; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Boyden Chamber Assay, Expressing, Western Blot, Injection, Imaging, Staining, Mutagenesis, Knockdown

Journal: Cancer Research

Article Title: ANO1 Reprograms Cholesterol Metabolism and the Tumor Microenvironment to Promote Cancer Metastasis

doi: 10.1158/0008-5472.can-22-3490

Figure Lengend Snippet: Figure 8. Working model of ANO1 promotes cancer metastasis by regulating CYP27A1–LXR signaling. ANO1 interacts with JUN to inhibit the transcription of CYP27A1 and inactivates LXR signaling, leading to increased cholesterol level and microenvironment reprogramming, therefore promoting cancer metastasis in ESCC. [The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license (https://smart.servier.com).]

Article Snippet: In brief, sections were blocked with 1% BSA in PBST buffer for 2 hours, and incubated with ANO1 antibody (Proteintech) overnight at 4 C (Supplementary Table S1).

Techniques: Generated

Journal: Scientific Reports

Article Title: Afatinib amplifies cAMP-induced fluid secretion in a mouse mini-gut model via TMEM16A-mediated fluid secretion and secretory cell differentiation

doi: 10.1038/s41598-025-14516-9

Figure Lengend Snippet: Effect of prolonged treatment with afatinib on expression of membrane transport proteins involved in chloride secretion. ( a ) Effect on mRNA expression. qRT-PCR was performed to measure mRNA expression of TMEM16A, CFTR, potassium calcium-activated channel subfamily N member 4 (K Ca 3.1), potassium voltage-gated channel subfamily Q member 1 (K v 7.1), and sodium potassium chloride cotransporter 1 (NKCC1), normalized by vehicle, after treatment of AFT (0.1 µM) or vehicle for 24 h ( n = 3–4). Western blot analyses were performed to investigate the protein expression of NKCC1 ( b ), K v 7.1( c ), and TMEM16A ( d ) after treatment with AFT or vehicle for 24 h. The blots were cropped for clarity, and the original images are provided in Supplementary Figure S3. Results are means ± S.E.M. ( n = 3 technical replicates). * , P < 0.05; ** , P < 0.01 compared with vehicle. All results were analyzed by Student t-test.

Article Snippet: Following membrane staining, colonoids were permeabilized with 0.3% Triton X-100 for 10 min and blocked with 1% bovine serum albumin (BSA) for 30 min. Then, they were incubated overnight at 4 °C with primary antibodies targeting TMEM16A (1:50 dilution, bs3794R, Bioss Inc., Woburn, MA, USA).

Techniques: Expressing, Membrane, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Afatinib amplifies cAMP-induced fluid secretion in a mouse mini-gut model via TMEM16A-mediated fluid secretion and secretory cell differentiation

doi: 10.1038/s41598-025-14516-9

Figure Lengend Snippet: Role of PI3K in afatinib-induced alteration of transport protein expression and secretory lineage differentiation. ( a ) Confirmation of PI3K inhibition by afatinib. Western blot analysis was performed to measure the protein expression of P-PI3K in colonoids treated with AFT (0.1 µM) or vehicle for 24 h. The blots were cropped for clarity, and the original images are provided in Supplementary Figure S4. Results are means ± S.E.M. ( n = 3). * , P < 0.05; ** , P < 0.01 compared with vehicle. The results were analyzed by Student t-test. ( b ) Role of PI3K in mediating the effect of afatinib on mRNA expression. Colonoids were treated with vehicle, AFT, BAY-80-6946 (50 nM; a PI3K inhibitor) or AFT plus BAY-80-6946 for 24 h before mRNA extraction and qRT-PCR being performed to analyze mRNA expression of NKCC1, K v 7.1, TMEM16A, ATOH1, and LYZ1. Results are means ± S.E.M. ( n = 3–7 technical replicates). * , P < 0.05; ** , P < 0.01 compared with vehicle. The results were analyzed by ANOVA followed by Turkey’s post hoc test.

Article Snippet: Following membrane staining, colonoids were permeabilized with 0.3% Triton X-100 for 10 min and blocked with 1% bovine serum albumin (BSA) for 30 min. Then, they were incubated overnight at 4 °C with primary antibodies targeting TMEM16A (1:50 dilution, bs3794R, Bioss Inc., Woburn, MA, USA).

Techniques: Expressing, Inhibition, Western Blot, Extraction, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Controlling the bioelectrical properties of neurons with ferritin-based Magnetogenetics

doi: 10.1101/2022.12.07.519516

Figure Lengend Snippet: ( A - D ) Electrical properties of N2a cells expressing TMEM16A FeRIC and TMEM16A WT were recorded in the voltage-clamp mode. Representative traces of whole-cell currents from N2a cells expressing ( A ) TMEM16A FeRIC or ( C ) TMEM16A WT in the baseline (gray) and within the 2 minutes after RF stimulation (post-RF, red). Average changes (±SEM) in Rm and Ra in the baseline and the post-RF period from N2a cells expressing ( B ) TMEM16A FeRIC or ( D ) TMEM16A WT . Average changes (±SEM) in YFP-H148Q (YFP) fluorescence in N2a cells expressing ( E ) TMEM16A FeRIC or ( G ) TMEM16A WT following the addition of imaging solution containing 70 mM iodide (black arrow) in the absence (black) or following RF stimulation (red). In a different set of experiments, cells were treated with the TMEM16A antagonist T16Ainh-A01 (T16Ainh, gray). Averages (±SEM) of the change in YFP fluorescence after 5 min of iodide application in N2a cells expressing ( F ) TMEM16A FeRIC or ( H ) TMEM16A WT for the different experimental groups. For imaging experiments, significance was determined using a one-way ANOVA followed by Bonferroni’s multiple comparisons tests. For Ephys experiments, significance was determined using a two-tailed Student’s t-test. Where applicable, either p < 0.05 (∗), p < 0.001 (∗∗), or p < 0.0001 (∗∗∗) was considered a statistically significant difference.

Article Snippet: To generate the TMEM16A WT construct, we used full-length TMEM16A cDNA, which was obtained from Origene (mouse untagged clone, PCMV6-Kan/Neo, #MC205263).

Techniques: Expressing, Fluorescence, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: Controlling the bioelectrical properties of neurons with ferritin-based Magnetogenetics

doi: 10.1101/2022.12.07.519516

Figure Lengend Snippet: ( A - D ) Membrane voltages recorded from neurons in the current-clamp mode. Representative traces of the membrane voltage in the baseline and within the 2 minutes after RF stimulation (post-RF) in neurons expressing TMEM16A FeRIC ( A ) in the absence or ( B ) in the presence of the antagonist Ani 9. ( C, D ) Average membrane potential (±SEM) before RF, during RF, and in the post-RF period in neurons expressing TMEM16A FeRIC ( C ) in the absence or ( D ) the presence of Ani 9. Insets: average membrane potential (±SEM) in the baseline and the post-RF period. ( E, F ) Spontaneous action potential firing in the baseline and the post-RF period in neurons expressing TMEM16A FeRIC ( E ) in the absence or ( F ) in the presence of Ani 9. Insets: number of action potentials (±SEM) in neurons expressing TMEM16A FeRIC in the baseline and the post-RF period. Significance was determined using a two-tailed Student’s t-test. Where applicable, p < 0.05 (∗) was considered a statistically significant difference.

Article Snippet: To generate the TMEM16A WT construct, we used full-length TMEM16A cDNA, which was obtained from Origene (mouse untagged clone, PCMV6-Kan/Neo, #MC205263).

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Controlling the bioelectrical properties of neurons with ferritin-based Magnetogenetics

doi: 10.1101/2022.12.07.519516

Figure Lengend Snippet: ( A ) Average changes (±SEM) in GCaMP6 fluorescence in neurons expressing TMEM16A FeRIC following the addition of 70 mM K + (black arrow) in non-stimulated (black), stimulated with RF (red) or stimulated with RF in the presence of Ani 9 (blue). ( B ) Bar graphs are the averages (±SEM) of the maximum change in GCaMP6 fluorescence (Max ΔF/F0) and the area under the curve (AUC) of neurons expressing TMEM16A FeRIC for the different experimental groups. ( C ) Representative changes in BeRST 1 fluorescence in neurons expressing TMEM16A FeRIC following treatment with bicuculline for 10 min in the absence (top) or the presence of RF stimulation (bottom). ( D ) Averages (±SEM) of the spiking frequency (Hz) for all mCherry+ neurons (spiking and non-spiking) for all the experimental groups. Experimental groups are independent samples. Baseline corresponds to the spike frequency in the absence of any stimulus. Neurons were imaged after treatment with bicuculline for 10 min in the absence or the presence of RF or RF plus Ani 9. ( K ) Pie charts of the fraction of mCherry+ neurons that were spiking (at least one spike in the entire experiment) or not spiking for the different experimental groups. Significance was determined using one-way ANOVA followed by Bonferroni’s multiple comparisons tests. Where applicable, either p < 0.05 (∗), p < 0.001 (∗∗), or p < 0.0001 (∗∗∗) was considered a statistically significant difference.

Article Snippet: To generate the TMEM16A WT construct, we used full-length TMEM16A cDNA, which was obtained from Origene (mouse untagged clone, PCMV6-Kan/Neo, #MC205263).

Techniques: Fluorescence, Expressing

Journal: Frontiers in Immunology

Article Title: DOG1 as a novel antibody-drug conjugate target for the treatment of multiple gastrointestinal tumors and liver metastasis

doi: 10.3389/fimmu.2023.1051506

Figure Lengend Snippet: DOG1 expression analysis at the gene and protein levels in tumor tissues and CTCs. (A) Venn diagram showing the overlaps between the overexpressed target sets for COAD: Colon adenocarcinoma, ESCA: Esophageal carcinoma, STAD: Stomach adenocarcinoma and MP: Membrane protein; (B) RNA-seq data of multiple gastrointestinal cancers from TCGA analyzed by UCSC Xena (the University of California, Santa Cruz) showed DOG1 RNA expression in primary tumors compared to normal tissues adjacent to the tumor. Axis units are log2 (normalized count+1). Student’s t test; (C) Representative immunohistochemical images for DOG1 protein expression in primary tumors. Positive IHC staining for DOG1 is indicated by a brown precipitate. Scale bar, 50 µm; (D) Representative IHC for DOG1in tissue samples from colon cancer patients with liver metastasis. Scar bar is 200 µm (left) and 100 µm (right); (E) DOG1 IHC scores of tissue samples from colon cancer patients with liver metastasis in (D–F) Immunoblot of DOG1 expression in tissue samples from colon cancer patients with liver metastasis by western blot. Quantitative analysis of DOG1 protein expression in tissue samples from colon cancer patients with liver metastasis by western blot (n=3). Commercial anti-DOG1 antibody sp31 used in (A-F) (G) Detection of DOG1+ CTCs from colon cancer patient by flow cytometry. Erythrocytes were lysed and cells were stained with an antibody cocktail against CD45, EpCam, Pan Cytokeratin (CK10, 14, 15, 16 and 19) and DOG1 (commercial antibody NBP2-34812AF405). EpCam + CK + CD45 − tumor cells were detected by flow cytometry by first gating out the cell debris and cell clumps in the forward/side scatter plot (gate A). Then, the CD45 + (FITC) cells were excluded by gating at the CD45 − cell population (gate B). The thresholds for specific EpCam (APC) and cytokeratin (PE) signals were determined using the sample stained with the isotype control antibodies (gate C). Gates were set to have no positive events above these thresholds in the control sample (gate C++); (H) The same gating strategy was then applied for detecting EpCam + CK + CD45 − cells in the sample stained with the specific antibodies. DOG1 + (PB450) cells were sellected in gate (D) In this plasma sample of colon cancer patient with liver metastasis, 100% (1/1) DOG1 + cells were selected out in CTCs; (I) Kaplan–Meier survival curves for patients with cardia adenocarcinoma, gastric adenocarcinoma, hepatocellular carcinoma and colon adenocarcinoma with DOG1+/− staining are shown. Log-rank test. Comparison within groups: *P < 0.05; **P <0.01; ***P <0.001; ****P <0.0001.

Article Snippet: The remaining cells were resuspended in 1 ml of staining buffer (0.5% bovine serum albumin (BSA), 2 mM EDTA in PBS), split into two equal fractions, and stained with specific antibodies against EpCam (APC-labeled, 324207-25, Biolegend), CD45 (Alexa Fluor 488-labeled, 53-9458-82, eBioscience) and DOG1 (Alexa Fluor 405-labeled, clone SPM580, NBP2-34812AF405, Novus Bio) or the relevant isotype control antibodies (BD Biosciences).

Techniques: Expressing, Membrane, RNA Sequencing Assay, RNA Expression, Immunohistochemical staining, Immunohistochemistry, Western Blot, Flow Cytometry, Staining, Control, Comparison

Journal: Frontiers in Immunology

Article Title: DOG1 as a novel antibody-drug conjugate target for the treatment of multiple gastrointestinal tumors and liver metastasis

doi: 10.3389/fimmu.2023.1051506

Figure Lengend Snippet: The positive expression rate of DOG1 in human tumor TMAs.

Article Snippet: The remaining cells were resuspended in 1 ml of staining buffer (0.5% bovine serum albumin (BSA), 2 mM EDTA in PBS), split into two equal fractions, and stained with specific antibodies against EpCam (APC-labeled, 324207-25, Biolegend), CD45 (Alexa Fluor 488-labeled, 53-9458-82, eBioscience) and DOG1 (Alexa Fluor 405-labeled, clone SPM580, NBP2-34812AF405, Novus Bio) or the relevant isotype control antibodies (BD Biosciences).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: DOG1 as a novel antibody-drug conjugate target for the treatment of multiple gastrointestinal tumors and liver metastasis

doi: 10.3389/fimmu.2023.1051506

Figure Lengend Snippet: DOG1 was expressed on the cell surface. (A) Representative graphs of DOG1 surface expression analyzed by flow cytometry. Blue is the isotype control group, and red is the anti-DOG1 antibody group; (B) Flow cytometric detection of DOG1 protein expression on the surface in various tumor cell lines, including GIST cells and colon, esophageal, liver and gastric cancer cells. Three independent experiments were performed; (C) DOG1 mRNA expression in various tumor cell lines, including GIST cells and colon, esophageal, liver and gastric cancer cells, were detected by qPCR. Three independent experiments were performed.

Article Snippet: The remaining cells were resuspended in 1 ml of staining buffer (0.5% bovine serum albumin (BSA), 2 mM EDTA in PBS), split into two equal fractions, and stained with specific antibodies against EpCam (APC-labeled, 324207-25, Biolegend), CD45 (Alexa Fluor 488-labeled, 53-9458-82, eBioscience) and DOG1 (Alexa Fluor 405-labeled, clone SPM580, NBP2-34812AF405, Novus Bio) or the relevant isotype control antibodies (BD Biosciences).

Techniques: Expressing, Flow Cytometry, Control

Journal: Frontiers in Immunology

Article Title: DOG1 as a novel antibody-drug conjugate target for the treatment of multiple gastrointestinal tumors and liver metastasis

doi: 10.3389/fimmu.2023.1051506

Figure Lengend Snippet: Anti-DOG1 antibody induced cell apoptosis and inhibited cell migration and invasion through p53 signaling pathway in HT-29 colon cancer cells. (A) The wound closure for HT-29 and GIST-882 was quantified at every 24 h post-wound (mean ± S.E.M., n = 6); (B) Representative image of wound healing assay in HT-29 cells at 0 h, 24 h and 48 h post wounding. The cells were treated with 100 nM and 200 nM anti-DOG1 antibody. Scale bar, 500 μm; (C) Migration (without Matrigel) and invasion (with Matrigel) of HT-29 cells were suppressed by the anti-DOG1 antibody compared with the control as shown by Transwell assays. Representative images are shown. Scale bar, 100 µm. Bar graphs of panel C are shown. Values are the mean ± SD; n=6; (D) Anti-DOG1 antibody-induced apoptosis in HT-29 cells. Apoptotic cells were quantified by Annexin V/PI double staining assay. HT-29 cells are treated with vehicle, 100 nM and 200 nM anti-DOG1 antibody for 48 h. Analysis on cell apoptosis results of I (n=3); (E) Flow cytometric analysis of the cell cycle distribution in HT-29 cells treated with vehicle, 100 nM and 200 nM anti-DOG1 antibody for 48 h. Bar graphs showing an increase of G1 phase and a decrease of S phase in cell cycle for the percentage of indicated cells in K (n=3), but without statistical significance; (F) Expression levels of 10 major cell signaling pathways in HT-29 cells treated with anti-DOG1 antibody. Values are the mean ± SD; n=3; (G) Relative mRNA-level of p53, Notch and TGFβ signaling of HT-29 cells after 48 h treatment with vehicle, 20 μM T16ainh-A01 and 200 nM anti-DOG1 antibody as determined by qRT-PCR. Data are normalized to the respective vehicle control and represent the mean ± SD; n=3; (H) Immunoblots of lysates from the HT-29 cell lines after 48 h treatment with vehicle, 20 μM T16ainh-A01, 100nM and 200 nM anti-DOG1 antibody. β-actin was used as control; (I) Bar graph showing quantitative analysis of protein expressions (n=3). Data were normalized by β-actin. Compared with the control group by one-way ANOVA. *P < 0.05; **P <0.01; ***P <0.001; ****P <0.0001.

Article Snippet: The remaining cells were resuspended in 1 ml of staining buffer (0.5% bovine serum albumin (BSA), 2 mM EDTA in PBS), split into two equal fractions, and stained with specific antibodies against EpCam (APC-labeled, 324207-25, Biolegend), CD45 (Alexa Fluor 488-labeled, 53-9458-82, eBioscience) and DOG1 (Alexa Fluor 405-labeled, clone SPM580, NBP2-34812AF405, Novus Bio) or the relevant isotype control antibodies (BD Biosciences).

Techniques: Migration, Wound Healing Assay, Control, Double Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Immunology

Article Title: DOG1 as a novel antibody-drug conjugate target for the treatment of multiple gastrointestinal tumors and liver metastasis

doi: 10.3389/fimmu.2023.1051506

Figure Lengend Snippet: DOG1 could mediate the internalization of anti-DOG1 antibodies. (A) The internalization rate of the anti-DOG1 antibody was calculated using the formula [1-MFItime/MFIcontrol]×100%. DOG1 on the cell surface was detected by flow cytometry (n=3); (B) Immunofluorescence microscopy observation of the endocytosis of Cy5.5-labeled anti-DOG1 DM4 ADC mediated by DOG1 protein in GIST882, HT-29, HepG2, and AGS cell lines. The results are from 0 h, 1 h, 4 h and 8 h. Antibodies were stained with Cy5.5, rhodamine-labeled phalloidin was used to visualize the actin cytoskeleton (green), and Hoechst (blue) was used for nuclear staining. Scale bar, 25 µm; (C) Bar graph showing quantitative analysis of protein expressions internalization rate of the immunofluorescence images (n=3). ***, P <0.001; ****, P <0.0001.

Article Snippet: The remaining cells were resuspended in 1 ml of staining buffer (0.5% bovine serum albumin (BSA), 2 mM EDTA in PBS), split into two equal fractions, and stained with specific antibodies against EpCam (APC-labeled, 324207-25, Biolegend), CD45 (Alexa Fluor 488-labeled, 53-9458-82, eBioscience) and DOG1 (Alexa Fluor 405-labeled, clone SPM580, NBP2-34812AF405, Novus Bio) or the relevant isotype control antibodies (BD Biosciences).

Techniques: Flow Cytometry, Immunofluorescence, Microscopy, Labeling, Staining

Journal: Frontiers in Immunology

Article Title: DOG1 as a novel antibody-drug conjugate target for the treatment of multiple gastrointestinal tumors and liver metastasis

doi: 10.3389/fimmu.2023.1051506

Figure Lengend Snippet: Anti-DOG1 ADCs showed potent in vitro and in vivo anti-tumor efficacy in multiple types of gastrointestinal tumor. (A) GIST882 and IM-resistant GIST882 cell lines were incubated with increasing concentrations of IM, unconjugated anti-DOG1 antibodies and anti-DOG1ADCs for 72 h. HT-29, HCT-116, LoVo, HepG2, HCC-LM3, MGC-803, and Kyse-410 cell lines were incubated with increasing concentrations of unconjugated anti-DOG1 antibodies and anti-DOG1 ADCs for 72 h. The cytotoxicity was calculated by IC 50 . NA: not active; (B) GIST PDX model (n=5); (C) HT-29 CDX model (n=5); (D) HepG2 CDX model (n=7); (E) MGC-803 CDX model (n=5); (F) Kyse-410 CDX model (n=5) were i.v. dosed Q3Dx3 as indicated (arrow) with vehicle, unconjugated anti-DOG1 antibodies at 10 mg/kg and anti-DOG1 ADCs at 5 or 10 mg/kg. Data from the tumor growth studies are depicted as the mean ± SEM. Compared with the control group by one-way ANOVA. ***P <0.001; ****P <0.0001. The body weight data are depicted as the mean ± SEM.

Article Snippet: The remaining cells were resuspended in 1 ml of staining buffer (0.5% bovine serum albumin (BSA), 2 mM EDTA in PBS), split into two equal fractions, and stained with specific antibodies against EpCam (APC-labeled, 324207-25, Biolegend), CD45 (Alexa Fluor 488-labeled, 53-9458-82, eBioscience) and DOG1 (Alexa Fluor 405-labeled, clone SPM580, NBP2-34812AF405, Novus Bio) or the relevant isotype control antibodies (BD Biosciences).

Techniques: In Vitro, In Vivo, Incubation, Control

Journal: Frontiers in Immunology

Article Title: DOG1 as a novel antibody-drug conjugate target for the treatment of multiple gastrointestinal tumors and liver metastasis

doi: 10.3389/fimmu.2023.1051506

Figure Lengend Snippet: Anti-DOG1 antibody inhibited the experimental liver metastasis model of colon cancer. (A) Timeline of drug administration of the treated mice; (B) Bioluminescence on Day 1 to 35 post-HT-29-Luc cell injection; (C) The change in body weight during the experiment was calculated as the percent change in weight compared with the baseline measurement. Values are the mean ± SEM; n =5 mice per group; compared with the control group by one-way ANOVA; (D) BrdU positive rate of liver tissue on Day 35. Values are the mean ± SEM; n=3 mice per group; compared with the control group by one-way ANOVA; (E) DOG1, HE and BrdU IHC staining for liver tissue on Day 35; (F) Representative images of liver in the HT-29-Luc-bearing mice on Day 35; (G) The graph depicted ALB, ALT, AST Tbil on Day 40 after the inoculation. Values are the mean ± SEM; n=3 mice per group; compared with the control group by Friedman test or Kruskal-Wallis test. *P < 0.05; ****P <0.0001.

Article Snippet: The remaining cells were resuspended in 1 ml of staining buffer (0.5% bovine serum albumin (BSA), 2 mM EDTA in PBS), split into two equal fractions, and stained with specific antibodies against EpCam (APC-labeled, 324207-25, Biolegend), CD45 (Alexa Fluor 488-labeled, 53-9458-82, eBioscience) and DOG1 (Alexa Fluor 405-labeled, clone SPM580, NBP2-34812AF405, Novus Bio) or the relevant isotype control antibodies (BD Biosciences).

Techniques: Injection, Control, Immunohistochemistry

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: Expression of TMEM16A in hepatocellular carcinoma and pericarcinous tissue. Notes: Expression of TMEM16A at ( A ) mRNA level and ( B ) protein level. ( C ) Quantification of the protein bands (OD ratio over GAPDH). Tumor: hepatocellular carcinoma; Normal: pericarcinous tissue. ** P <0.01. Abbreviations: OD, optical density; N, normal tissues; T, tumor tissues.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: Expression of TMEM16A in SMMC-7721 cells after TMEM16A siRNA transfection. Notes: ( A ) qRT-PCR analyzed the mRNA expression of TMEM16A in SMMC-7721 cells after TMEM16A-siRNA transfection. ( B ) The TMEM16A protein expression level in SMMC-7721 cells after TMEM16A-siRNA transfection was detected using Western blot. Control: normal SMMC-7721 cells; siRNA: cells transfected with TMEM16A siRNA. ** P <0.01 vs control. Abbreviation: NC, negative control.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Negative Control

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: The proliferation, migration, and invasion of SMMC-7721 cells were attenuated by knockdown of TMEM16A. Notes: ( A ) After transfection, the cell proliferation was detected by MTT assay. ( B ) The migration and invasion were assessed by transwell chamber, and ( C ) migrated cells were counted. Control: normal SMMC-7721 cells; NC: negative control; siRNA: cells transfected with TMEM16A siRNA. * P <0.05 and ** P <0.01 vs control. Abbreviation: h, hours.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Migration, Transfection, MTT Assay, Negative Control

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: Cell cycle and cell apoptosis of SMMC-7721 cells that were transfected with TMEM16A siRNA. Notes: ( A ) Cell cycle distribution was measured by flow cytometry with cell cycle staining kit and ( B ) the cell cycle phase is shown in a bar graph with the G0/G1, S, and G2/M phases. ( C ) Cell apoptosis in each group was determined using the Annexin V-FITC/PI flow cytometry, and ( D ) proportion of apoptosis cells was measured. Control: normal SMMC-7721 cells; NC: negative control; siRNA: cells transfected with TMEM16A siRNA. * P <0.05 vs control. Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide; V-FITC/PI, fluorescein isothiocyanate-conjugated Annexin V and PI.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Transfection, Flow Cytometry, Staining, Negative Control

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: Expression of MAPK signaling proteins (p38, p-p38, ERK1/2, p-ERK1/2, JNK, and p-JNK) and cell cycle regulatory protein cyclin D1 in TMEM16A siRNA-transfected SMMC-7721 cells. Abbreviation: NC, negative control.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: Expressing, Transfection, Negative Control

Journal: OncoTargets and therapy

Article Title: Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

doi: 10.2147/OTT.S95985

Figure Lengend Snippet: Knockdown of TMEM16A suppresses tumorigenicity in vivo. Notes: Tumor volume ( V ) was measured daily by caliper and was calculated using the formula V = ( L × W 2 )/2, where L was the length and W was the width of the tumor. Growth curves were plotted using average tumor volume within each experimental group every week after inoculation of TMEM16A shRNA-transfected SMMC-7721 cells and NC shRNA-transfected SMMC-7721 cells. Six weeks later, the mice were euthanized, and ( A ) the dissected tumors were collected and measured (in cm), and ( B ) the tumor growth curves were determined. * P <0.05 siRNA vs NC. Abbreviation: NC, negative control.

Article Snippet: For knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, USA).

Techniques: In Vivo, shRNA, Transfection, Negative Control

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 1. Pharmacological block or genetic knockdown of ANO1 produces a similar inhi- bition of the contraction of mouse pulmonary artery to 5-HT as blocking VGCC or emptying Ca2+ stores from the SR. (A) Typical isometric force recordings in response to high K+ Krebs (85.4 mM) and increasing cumulative concen- trations of 5-HT ranging from 0.01 to 30 μM as indicated by the bars above the traces, in the absence (left) or presence (right) of the ANO1 inhibitor CaCCInh-A01, also indicated by a hori- zontal bar above the trace. (B) Mean cumulative dose–response curves to 5-HT in mouse pul- monary arteries from wild-type C57/BL6 mice in the absence (black circles, Control; n = 14), or presence (blue squares; n = 5) of 1 μM nifedipine to block VGCC, 10 μM CPA to deplete SR Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Blocking Assay, Knockdown, Control

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 2. The ANO1 blocker CaCCInh-A01 produced no effect on the high K+-mediated contraction of the mouse pulmonary artery. (A) Typical contractile force experiment showing that increasing the concentration of CaCCInh-A01 from 1 to 30 μM (progressively thickening black bar shown over the trace) produced no notice- able effect on the contraction (blue trace) eli- cited by 85.4 mM K+–Krebs solution (K+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Produced, Concentration Assay

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 3. Ca2+ oscillations triggered by 5-HT in individual smooth muscle cells from an intact mouse endothelium-denuded PA are potently inhibited by the inhibition of ANO1. All data were collected from the same PA from a conditional smooth muscle–specific and inducible GCaMP3 mouse injected with tamoxifen to induce Cre expression. (A) Ca2+ imaging was performed in the absence of an agonist (Control). The left panel shows one image from a video from which a ST map (middle colored image) was created in the area spanned by the diagonal white line. Fluorescence intensity was measured under the three white lines on the ST map (corresponding to two different cells) and plotted as a function of time as shown on the right. There was no detectable activity in these two cells as well as across the entire field of view of the movie. (B) Same nomenclature as in A except that the preparation was exposed to 1 μM 5-HT for 5 min. A ST map created in the same manner as that in A shows clear evidence of asynchronous Ca2+ transients. This is more evident from examining the fluorescence intensity profile of the same two cells analyzed in A, which displayed repetitive Ca2+ transient of distinct magnitude and frequency. (C) The nomenclature of this panel is identical to that of B and C, with the exception that the PA was exposed to 10 μM CaCCInh-A01 for 10 min while still being incubated with 5-HT. Examination of the ST map reveals little, if any, Ca2+ oscillations in the presence of the ANO1 inhibitor; Ca2+ transients were no longer apparent in the same two cells analyzed in A and B.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Inhibition, Injection, Expressing, Imaging, Control, Fluorescence, Activity Assay, Incubation

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 5. Sample experiment illustrating how ANO1 knockdown exerted a strong inhibition of 5-HT-induced Ca2+ oscillations in a PA from a tamoxifen-injected SMC-ANO1-KO-ΔEx12-GCaMP3 mouse. The top left panel is an image from a video stack recorded in a pulmonary artery from a con- ditional smooth muscle cell-specific and inducible ANO1 knockout mouse expressing GCaMP3 specifically in smooth muscle cells, which was exposed to 1 μM 5- HT for 5 min. One ST map constructed from the white line crossing the image is shown in the lower left corner and reveals very little activity. The fluorescence intensity profile as a function of time of two cells from the ST map labeled with the letters a and b are shown on the right. Cell 1 displayed no significant Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Knockdown, Inhibition, Injection, Knock-Out, Expressing, Construct, Activity Assay, Fluorescence, Labeling

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 6. Asynchronous Ca2+ oscillations evoked by 5-HT require both functional ANO1 and VGCC. Mean data for each of four parameters measured from Ca2+ transients elicited by 1 μM 5-HT (5 min) in PA from control SMC-GCaMP3 (light blue bars) or SMC-ANO1-KO-ΔEx12-GCaMP3 (light gray bars) mice. (A–D) The frequency of Ca2+ oscillations (A), peak Ca2+ transient amplitude (F/F0; B), integrated area under the curve (C), and FWHM (D) were measured as shown in the upper right corner. For each dataset, the mean is indicated by a filled black square with the colored boxes and whiskers delimiting the 25th and 75th percentile, and the 10th and 90th percentile of the pooled data, respectively, and small dots individual data points. N: number of animals; n: number of cells. SMC-GCaMP3 + 5-HT: N = 7, n = 114 for peak, area under the curve, and FWHM, and n = 116 for frequency; SMC-GCaMP3 + 5-HT + CaCCInh-A01 (CaCCInh): N = 7, n = 15 for peak, area under the curve, and FWHM, and n = 76 for frequency; SMC-GCaMP3 + 5-HT + nifedipine (Nif): N = 2, n = 32 for peak, area under the curve, and FWHM, and n = 47 for frequency; GCaMP3 + 5-HT + CPA: N = 2, n = 29; SMC-ANO1-KO-ΔEx12-GCaMP3; 5-HT: N = 7, n = 39 for peak, area under the curve, and FWHM, and n = 137 for frequency. For all panels, ***, **, and * indicate a significant difference between means with P < 0.001, P < 0.01, and P < 0.05, respectively.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Functional Assay, Control

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 7. Blocking ANO1 or CaV1.2 depletes SR Ca2+ stores. (A) Typical isometric force re- cording obtained under control conditions showing the effect of depleting the SR Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Blocking Assay, Control

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 8. ANO1, CaV1.2, and IP3R colocalize in peripheral coupling sites to form signaling complexes. (A and B) Co-IP of CaV1.2 or IP3R with ANO1 from lysates of the pulmonary artery from wild-type mice. Pulldown was carried out with anti-ANO1 antibody and then probed by Western blot with anti-CaV1.2, anti-IP3R, or anti- ANO1 antibodies. Five to six mouse tissues per experiment, each ran in triplicates. (C and D) Freshly isolated PASMCs from wild-type mice were immunolabeled for ANO1 and CaV1.2 (C) or ANO1 and IP3R (D). All three proteins were preferentially localized to the periphery of the cells. (D and F) Line profiles of the areas indi- cated by the white dashed lines in C and E. The fluorescence intensity was normalized to the minimum and maximum fluorescence for each sample. The black arrowheads denote the loca- tion of the PM. ANO1 and CaV1.2 show strong immunolabeling at the PM (D). (E) IP3R shows some intracellular immunolabeling, with moder- ate peaks present at the periphery showing an enhancement of protein localization to periphe- ral coupling sites. Source data are available for this figure: SourceData F8.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Isolation, Immunolabeling, Fluorescence

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 9. Superresolution imaging of ANO1, CaV1.2, and IP3R at the PM of PASMCs from wild-type mice. (A and B) Superresolution images of PASMCs labeled for ANO1 and CaV1.2 (A) or ANO1 and IP3R (B) were imaged using GSDIM in epifluorescence mode. Epifluorescence images are shown in the inset for

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Imaging, Labeling

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 10. Membrane cholesterol depletion with MβCD causes the internalization of ANO1 and CaV1.2 proteins. (A and C) Freshly isolated PASMCs from wild-type mice were im- munolabeled for ANO1 and CaV1.2 before (A) or after (C) a 30-min exposure to MβCD (3 mg/ml; MβCD) to deplete membrane cholesterol and disrupt lipid rafts. The two ion channel proteins were preferentially localized to the periphery of the cells in control conditions as similarly shown in Fig. 8. (B–D) Line profiles of the areas indi- cated by the white dashed lines in A and C are respectively displayed in B and D. For these plots, the fluorescence intensity was normalized to the minimum and maximum fluorescence for each sample. The black arrowheads denote the location of the PM. ANO1 and CaV1.2 show strong immunolabeling at the PM in control condition (C) and translocation toward the cen- ter core of the cell after exposure to MβCD (D). The cells from A and C were isolated from the same mouse. (E and F) Graphs summarizing the effects of exposing PASMCs to MβCD on the distribution of ANO1 (magenta bars) and CaV1.2 (green bars), respectively. Measurements were performed as described in the text and consisted in normalizing membrane fluorescence to total cell fluorescence. For each dataset, the mean is indicated by a large, filled black square with the colored boxes and whiskers delimiting the 25th and 75th percentile, and the 10th and 90th percentile of the pooled data, respectively, and small dots individual data points. N: number of animals; n: number of cells; for the control group (E): ANO1 and CaV1.2: N = 3, n = 43; for the MβCD group (F): ANO1 and CaV1.2: N = 3, n = 35. *** indicates a significant difference between means with P < 0.001.

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Membrane, Isolation, Control, Fluorescence, Immunolabeling, Translocation Assay

Journal: The Journal of general physiology

Article Title: ANO1, CaV1.2, and IP3R form a localized unit of EC-coupling in mouse pulmonary arterial smooth muscle.

doi: 10.1085/jgp.202213217

Figure Lengend Snippet: Figure 12. Hypothetical models of EC coupling involving ANO1, CaV1.2, and IP3R during agonist-mediated contraction of mouse pulmonary ar- terial smooth muscle cells. (A) General uniform model depicting the acti- vation of ANO1 by both Ca2+ release from IP3-sensitive SR Ca2+ stores and Ca2+ entry through CaV1.2. In this model, the three ion transporters are evenly distributed in the membrane and are not physically coupled. The depolari- zation is maintained by the positive feedback loop established by CaV1.2- mediated activation of Cl−efflux through ANO1 and its impact on the state of activation of CaV1.2 through regulation of membrane potential. (B) Schematic diagram illustrating the local interaction of ANO1, CaV1.2 with IP3R and their impact on membrane potential, Ca2+ entry, and contraction. In this model, the three ion channels are physically coupled in a restricted number of sites (Super Cluster) distributed across the long axis of the cell (shown as red boxes in the bottom diagram) and are organized for compartmentalized Ca2+

Article Snippet: Cells were incubated overnight at 4°C with primary antibodies: rabbit antibody to ANO1 (PA2290, 1:50; Boster Biological), mouse antibody to CaV1.2 (N263/31, 1:100; NeuroMab), or mouse antibody to IP3R (sc-377518, 1:100; Santa Cruz).

Techniques: Membrane, Activation Assay

Journal: Molecular medicine reports

Article Title: TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway.

doi: 10.3892/mmr.2016.4979

Figure Lengend Snippet: Figure 2. Decreased expression of TMEM16A, p‑MLC/MLC and p‑MYPT1/MYPT1 in rat basilar arteries during the development of hypertension. (A) Western blot analysis of TMEM16A in basilar arteries at various time‑points after 2‑kidney, 2‑clip surgery. Western blot analysis of phosphorylation of (B) MLC and (C) MYPT1 in basilar arteries. GAPDH served as a loading control. *P<0.05, **P<0.01 vs. week 0 (n=6). TMEM16A, transmembrane protein 16A; MLC, myosin light chain; MYPT1, myosin phosphatase‑targeting subunit 1; p, phosphorylated.

Article Snippet: Rabbit polyclonal TMEM16A antibody was obtained from Novus Biologicals, LLC (Littleton, CO, USA; cat. no. NBP1‐60076; dilution, 1:500).

Techniques: Expressing, Western Blot, Phospho-proteomics, Control

Journal: Molecular medicine reports

Article Title: TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway.

doi: 10.3892/mmr.2016.4979

Figure Lengend Snippet: Figure 3. Ang II evoked a TMEM16A‑mediated current using 100 nM [Ca2+]i in BASMCs. (A) Representative traces of ICl.Ca recorded in BASMCs. Cells were immersed in a bath solution and the current was recorded using a whole‑cell patch clamp with (a) a basal intracellular calcium concentration (100 nM [Ca2+]i) and (b) immediately after 100 nM Ang II was perfused into the bath solution. Results from BASMCs pretreated with (c) TMEM16A siRNA and 100 nM [Ca2+]i, 100 nM Ang II‑induced current for 48 h or (d) 10 µM losartan for 15 min. (e) Bar graph of current density at 100 mV in the different groups. (B) IClCa was recorded in BASMCs with (a) 500 nM [Ca2+]i and (b) the current wasn't enhanced by 100 nM Ang II. (c) Bar graph of current density at 100 mV in the two groups. *P<0.05, **P<0.01 (n=8‑11). Ang II, angiotensin II; TMEM16A, transmembrane protein 16A; [Ca2+]i, intracellular Ca2+; BASMC, basilar artery smooth muscle cell; IClCa, Ca2+‑dependent Cl− channel.

Article Snippet: Rabbit polyclonal TMEM16A antibody was obtained from Novus Biologicals, LLC (Littleton, CO, USA; cat. no. NBP1‐60076; dilution, 1:500).

Techniques: Patch Clamp, Concentration Assay

Journal: Molecular medicine reports

Article Title: TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway.

doi: 10.3892/mmr.2016.4979

Figure Lengend Snippet: Figure 4. Effect of si‑TMEM16A and adenovirus on TMEM16A expression in BASMCs. BASMCs were treated with (A) si‑TMEM16A or (B) adv‑TMEM16A for 3 and 6 h, respectively, and then cultured with 10% fetal bovine serum and Dulbecco's modified Eagle's medium/Ham's F-12 medium for another 48 h. TMEM16A protein expression was detected by western blot analysis and GAPDH served as a loading control. *P<0.05 vs. Neg Ctrl. n=4. TMEM16A, transmembrane protein 16A; BASMC, basilar artery smooth muscle cell; Neg Ctrl, negative control; si‑TMEM16A, TMEM16A siRNA; adv‑TMEM16A, TMEM16A adenovirus.

Article Snippet: Rabbit polyclonal TMEM16A antibody was obtained from Novus Biologicals, LLC (Littleton, CO, USA; cat. no. NBP1‐60076; dilution, 1:500).

Techniques: Expressing, Cell Culture, Modification, Western Blot, Control, Negative Control

Journal: Molecular medicine reports

Article Title: TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway.

doi: 10.3892/mmr.2016.4979

Figure Lengend Snippet: Figure 5. Regulation of Ang II‑induced phosphorylation of MLC and MYPT1 by TMEM16A expression. BASMCs were treated with (A and B) si‑TMEM16A or (C and D) adv‑TMEM16A for 3 h or 6 h and cultured for another 48 h. Y‑27632 (10 µM) was added 10 min before the 5‑min treatment of 100 nM Ang II. Cell lysates were collected and phosphorylation of (A and C) MLC and (B and D) MYPT1 were detected by western blot analysis. GAPDH served as a loading control. *P<0.05; n=8. Ang II, angiotensin II; MLC, myosin light chain; MYPT1, myosin phosphatase‑targeting subunit 1; TMEM16A, transmembrane pro tein 16A; si, small interfering; si‑TMEM16A, TMEM16A siRNA; BASMC, basilar artery smooth muscle cell; p, phosphorylated; adv‑TMEM16A, TMEM16A adenovirus.

Article Snippet: Rabbit polyclonal TMEM16A antibody was obtained from Novus Biologicals, LLC (Littleton, CO, USA; cat. no. NBP1‐60076; dilution, 1:500).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Western Blot, Control

Journal: Molecular medicine reports

Article Title: TMEM16A contributes to angiotensin II-induced cerebral vasoconstriction via the RhoA/ROCK signaling pathway.

doi: 10.3892/mmr.2016.4979

Figure Lengend Snippet: Figure 6. Expression of GTP‑RhoA/RhoA in cultured BASMCs in response to 100 nM Ang II following TMEM16A downregulation or overexpression. BASMCs were treated with (A) si‑TMEM16A or (B) adv‑TMEM16A for 3 h or 6 h and then cultured with 10% fetal bovine serum and Dulbecco's modified Eagle's medium/Ham's F-12 medium for another 48 h. AngII (100 nM) was added for 5 min to induce cell contraction. Cell lysates were collected and GTP‑RhoA and RhoA were detected by western blot analysis. GAPDH served as a loading control. *P<0.05; n=8. GTP, guanosine-5'-triphosphate; BASMC, basilar artery smooth muscle cell; TMEM16A, transmembrane protein 16A; si, small interfering; si‑TMEM16A, TMEM16A siRNA; adv‑TMEM16A, TMEM16A adenovirus; Neg Ctrl, negative control.

Article Snippet: Rabbit polyclonal TMEM16A antibody was obtained from Novus Biologicals, LLC (Littleton, CO, USA; cat. no. NBP1‐60076; dilution, 1:500).

Techniques: Expressing, Cell Culture, Over Expression, Modification, Western Blot, Control, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Endogenous Ca 2+ -dependent TMEM16A and cAMP-activated CFTR Cl − transport in HT 29 colonic epithelial cells. ( A , B ) YFP fluorescence quenching by iodide is enhanced by ATP in a concentration-dependent manner ( n = 5 for all). siRNA knockdown of TMEM16A but not TMEM16F inhibits quenching. ( C ) Semiquantitative RT-PCR indicates knockdown of TMEM16A (T16A; 92%, n = 3) and TMEM16F (T16F; 93%, n = 3). ( D , E ) Western blotting indicating pronounced expression of endogenous TMEM16A and CFTR in HT 29 cells. siRNA knocked down expression of TMEM16A. ( F – H ) Activation of chloride conductance by IBMX and forskolin (I/F; 100 µM/2 µM) was not detected in iodide quenching ( n = 5), but was significant in whole cell patch clamp recordings (overlay currents and I/V curves; n = 5). Mean ± SEM. * significant activation ( p < 0.05; paired t -test). # significant inhibition ( p < 0.05; ANOVA).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Fluorescence, Concentration Assay, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Activation Assay, Patch Clamp, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Eact does not activate endogenous Ca 2+ -dependent Cl − secretion, and has little effect on [Ca 2+ ] i . ( A , B ) No activation of iodide quenching by Eact or GSK1016790 ( n = 5 for both). ( C ) RT-PCR indicating the expression of TRPV4 in HT 29 cells. ( D , E ) No activation of whole cell currents in HT 29 cells by Eact or GSK1016790 (10 µM; n = 5 for both). ( F , G ) RT-PCR of TRPV4 expressed in CFBE ( F ) and HEK293 ( G ) cells. ( H ) ATP (100 µM) induced [Ca 2+ ] i rise is inhibited by knockdown of TMEM16A but not by Ani9 (10 µM; n = 37–105). ( I , J ) Minor increase in [Ca 2+ ] i by Eact in HT 29 , CFBE, or HEK293 cells ( n = 31–111). Mean ± SEM. # significant inhibition ( p < 0.05; unpaired t -test).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Knockdown, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Effect of inhibitors on Ca 2+ -activated Cl − transport and ATP-induced rise in [Ca 2+ ] i . A ) Increase in intracellular Ca 2+ by stimulation of HT 29 cells with ATP (100 µM). In contrast to benzbromarone and niclosamide, Ani9 did not attenuate the effect of ATP on [Ca 2+ ] i ( n = 7–23). In Call33 head and neck cancer cells and M1 mouse collecting duct cells Ani9 (10 µM) inhibited ATP-induced Ca 2+ increase ( n = 134–162) significantly. B ) Inhibition of ATP (5 µM) activated YFP-quenching in HT 29 cells by BBR (10 µM). C ) Inhibition of ionomycin (Iono, 1 µM) activated whole cell currents by BBR in HEK293 cells overexpressing TMEM16A (original recording and I/V curves) ( n = 5–6). D ) Inhibition of ionomycin (Iono; 1 µM) activated whole cell currents by BBR in HEK293 cells overexpressing TMEM16F ( n = 6, original recording and I/V curves). E ) Inhibition of ATP (5 µM) induced YFP-quenching in HT 29 cells by Ani9. F , G ) Inhibition of ionomycin (Iono; 1 µM) activated TMEM16A currents by Ani9 (10 µM), and change in time-dependent activation of TMEM16F currents ( n = 5–8). * significant inhibition ( p < 0.05; paired t -test). # significant inhibition ( p < 0.05; unpaired t -test).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Inhibition, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Niclosamide inhibits TMEM16A currents activated by intracellular Ca 2+ . (A ) TMEM16A overexpressed in HEK293 cells was activated by 1 µM Ca 2+ in the patch pipette filling solution. Acute application of niclosamide (Niclo; 5 µM) significantly inhibited Ca 2+ -activated TMEM16A whole cell currents ( n = 7). ( B ) 15 min preincubation with Niclo inhibited TMEM16A more potently ( n = 9–10). ( C ) Increase in intracellular Ca 2+ with ATP (100 µM) in HT 29 cells. Niclosamide (5 µM) induces a slight and transient increase in [Ca 2+ ] i and inhibits ATP-induced rise in [Ca 2+ ] i . siRNA knockdown of TMEM16A inhibits increase in [Ca 2+ ] i by ATP. Niclosamide shows no additional effects on [Ca 2+ ] i ( n = 60–193). ( D ) ER Ca 2+ store release and Ca 2+ influx (SOCE) induced by CPA and niclosamide ( n = 40–50). ( E , F ) Effects of CPA and niclosamide on Ca 2+ store release and SOCE under various conditions ( n = 40–213) * significant inhibition ( p < 0.05; paired t -test). # significant inhibition ( p < 0.05; unpaired t -test).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Transferring, Knockdown, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Endogenous and overexpressed TMEM16A behave differently. ( A ) Activation of overexpressed TMEM16A whole cell currents in HEK293 cells by Eact (10 µM, n = 9). ( B ) Activation of TMEM16F whole cell currents in TMEM16F-overexpressing HEK293 cells by Eact (10 µM, n = 6). ( C , D ) Little activation of endogenous TMEM16A currents by melittin (200 nM; n = 10) in HT29 cells, but strong activation in HEK293 cells overexpressing TMEM16A ( n = 7). ( E , F ) Little activation of endogenous TMEM16A currents by cinnamaldehyde (Cinna; 1 µM; n = 9), but strong activation of overexpressed TMEM16A ( n = 5). ( G , H ) Little activation of endogenous TMEM16A currents by diC8-PIP 2 (50 µM; n = 6), but strong activation of overexpressed TMEM16A ( n = 6). * significant activation ( p < 0.05; paired t -test). # significant activation ( p < 0.05; unpaired t -test).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: Effects of potential activators/potentiators of TMEM16A in HT29 cells (endogenous TMEM16A) and HEK293 cells (overexpressed TMEM16A).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: diC8-PIP2 augments TMEM16A currents activated by ionomycin. ( A – D ) Whole cell currents and I/V curves showing effect of diC8-PIP 2 (50 µM in the patch pipette filling solution) on basal and ionomycin (Iono, 0.1 µM) activated TMEM16A currents in HT 29 cells ( A , B ) and HEK293 cells ( C , D ). Activation of TMEM16A by diC8-PIP2 is clearly observed in TMEM16A-overexpressing HEK293 cells but not in HT 29 cells ( n = 6–7 for all). ( E , F ) Time courses for Iono-activated TMEM16A currents in HT 29 and HEK293 cells ( n = 6–8). * significant activation ( p < 0.05; paired t -test). # significant difference to the absence of diC8-PIP 2 ( p < 0.05; unpaired t -test).

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: Transferring, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition and Activation of the Ca 2+ Activated Cl − Channel TMEM16A

doi: 10.3390/ijms21072557

Figure Lengend Snippet: RT-PCR primers.

Article Snippet: Membranes were incubated with primary rabbit polyclonal anti-TMEM16A DOG1 antibody (#NBP1-49799, Novus Biologicals, Centennial, CO, USA; 1:500 in 1% ( w/v ) NFM/TBS-T) overnight at 4 °C.

Techniques: