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: 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: 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, RNA Expression, Immunohistochemical staining, Immunohistochemistry, Western Blot, Flow Cytometry, Staining, Control, Clinical Proteomics, 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, Protein-Protein interactions, 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: 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:

Journal: Biochemistry and Biophysics Reports

Article Title: Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

doi: 10.1016/j.bbrep.2021.100912

Figure Lengend Snippet: Ano1 mRNA expressed differentially and carbachol (CCH) stimulates Isc both in mouse small and large intestine. (A) Represents the quantity of Ano1 gene expression. Data are means ± S.E (n = 3). (B) CCH stimulated Isc in different parts of the mouse intestine, as indicated. *P < 0.01 (ANOVA with Bonferroni's test). (C) Effects of MONNA on basal and CCH-stimulated Isc and (D) representative tracing of apical and serosal incubation of MONNA in response to CCH stimulation in mouse colonic tissue. Data are means ± S.E (n = 4–7). (E) Showing representative Isc response to CCH in the presence of apical to serosal and serosal to apical Cl − gradient. Inset indicates the direction of the Cl − gradient. Ap, apical and Bl, serosal side of the mouse colon (n = 3).

Article Snippet: Mouse intestinal tissue sections were fixed in 3% paraformaldehyde prior to paraffin embedding [ , ].Thereafter, sections were incubated with rabbit anti -TMEM16A (Novus biological, #NBP2-29662) and mouse anti -NHERF1(Santa Cruz Biotechnology, #sc-271552) antibody overnight at 4 °C followed by exposure to goat anti-rabbit- IgG Alexa Fluor 488 or goat anti-mouse-IgG Alexa fluor 680 secondary antibody (Invitrogen; 1:500) for 1h at room temperature.

Techniques: Gene Expression, Incubation

Journal: Biochemistry and Biophysics Reports

Article Title: Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

doi: 10.1016/j.bbrep.2021.100912

Figure Lengend Snippet: Ano1 ± mouse caused reduction of basal and CCH-stimulated Isc. (A) Basal and CCH-stimulated Isc in the small intestine (jejunal) and colonic tissue of WT and Ano1 ± mice. Data are means ± S.E (n = 6).*P < 0.05. (B) Showing FSK-stimulated Isc response in WT and Ano1 ± mouse colonic mucosa. Data are means ± S.E (n = 4–6). (C) Representative western blot image of TMEM16A protein in different parts of wild type and Ano1 ± enterocytes. (D) Quantitative analysis of the immunoblots was determined by ImageJ analysis. Data are means ± S.E of three independent experiments. (* denotes significant difference at p value < 0.05, NS, non significant at p < 0.05).

Article Snippet: Mouse intestinal tissue sections were fixed in 3% paraformaldehyde prior to paraffin embedding [ , ].Thereafter, sections were incubated with rabbit anti -TMEM16A (Novus biological, #NBP2-29662) and mouse anti -NHERF1(Santa Cruz Biotechnology, #sc-271552) antibody overnight at 4 °C followed by exposure to goat anti-rabbit- IgG Alexa Fluor 488 or goat anti-mouse-IgG Alexa fluor 680 secondary antibody (Invitrogen; 1:500) for 1h at room temperature.

Techniques: Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

doi: 10.1016/j.bbrep.2021.100912

Figure Lengend Snippet: TMEM16A localizes to the apical membrane of mouse enterocytes and immunoprecipitates with NHERF1 in co-transfected HEK293T cells. (A) Representative western blot showing NHERF1 expression in mouse intestinal tissue as indicated. The histogram showing the densitometric quantification of NHERF1 protein expression, right to the immunoblot. This experiment was performed in triplicate. (B) Immunofluorescence demonstrated TMEM16A (green) and NHERF1 (red) in mouse colon, the yellow signal indicated co-localization of TMEM16A with NHERF1 (merged). Representative of two independent experiments. (C) TMEM16A co-immunoprecipitates with Flag-NHERF1 in co-transfected HEK293T cells. TMEM16A was detected only in the Co-IP complex from cell co-transfected with plasmids expressing both mouse Ano1 and Flag-Nherf1. (D) Depicted a reduced TMEM16A abundance in Co-IP complex from cell lysates with truncated (delC4) Ano1 compared to the result obtained with the control (full length). Representative blot of three independent experiments for (C) and (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Mouse intestinal tissue sections were fixed in 3% paraformaldehyde prior to paraffin embedding [ , ].Thereafter, sections were incubated with rabbit anti -TMEM16A (Novus biological, #NBP2-29662) and mouse anti -NHERF1(Santa Cruz Biotechnology, #sc-271552) antibody overnight at 4 °C followed by exposure to goat anti-rabbit- IgG Alexa Fluor 488 or goat anti-mouse-IgG Alexa fluor 680 secondary antibody (Invitrogen; 1:500) for 1h at room temperature.

Techniques: Membrane, Transfection, Western Blot, Expressing, Immunofluorescence, Co-Immunoprecipitation Assay, Control

Journal: Biochemistry and Biophysics Reports

Article Title: Intestinal TMEM16A control luminal chloride secretion in a NHERF1 dependent manner

doi: 10.1016/j.bbrep.2021.100912

Figure Lengend Snippet: Functional involvement of NHERF1 in TMEM16A mediated Cl − secretion. (A) NHERF1 was co-immunoprecipitated with TMEM16A endogenously from T84 cell lysates. No signal was detected using protein-G-conjugated beads alone (IP control). Representation of three independent experiments. ( B ) Western blot showing NHERF1 expression in wild type T84 (T84WT), vector control (T84VC) and NHERF1 knocked down cells (T84NF1KD736). (C) Histogram showing densitometric quantification of NHERF1 expression, right to the immunoblot. Result represents means ± S.E (n = 3). (D ) Representative traces showing the response of T84NF1KD736 cells to CCH-stimulated Isc. Inset showing summarized data from six independent experiments. Data are means ± S.E. (E) Effect of FSK pre-stimulation on CCH-stimulated Isc in WT and NHERF1KD T84 cell monolayers. Monolayers grown on filters were exposed to FSK (10 μM) and then stimulated with serosal CCH (100 μM). Data are mean ± SEM from 4 to 6 monolayers of each condition.

Article Snippet: Mouse intestinal tissue sections were fixed in 3% paraformaldehyde prior to paraffin embedding [ , ].Thereafter, sections were incubated with rabbit anti -TMEM16A (Novus biological, #NBP2-29662) and mouse anti -NHERF1(Santa Cruz Biotechnology, #sc-271552) antibody overnight at 4 °C followed by exposure to goat anti-rabbit- IgG Alexa Fluor 488 or goat anti-mouse-IgG Alexa fluor 680 secondary antibody (Invitrogen; 1:500) for 1h at room temperature.

Techniques: Functional Assay, Immunoprecipitation, Control, Western Blot, Expressing, Plasmid Preparation

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Epinephrine stimulation of anion secretion in the Calu-3 serous cell model.

doi: 10.1152/ajplung.00190.2013

Figure Lengend Snippet: Fig. 9. Localization of TMEM16A in Calu-3 cells. Wheat germ agglutinin (WGA) 555 (red) stain was used to indicate the cell boundaries including the apical membrane. Orthogonal sections from the confocal image stack show apical localization in monolayers stained with the Novus and the Aviva anti-TMEM16A antibodies (green). Insets: from the yellow lines as indicated in left and center panels.

Article Snippet: Anti-TMEM16A antibodies were purchased from Novus Biologicals (Littleton, CO) and Aviva Systems Biology (#ARP42506; San Diego, CA).

Techniques: Staining, Membrane

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