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: 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 tumorigenicity assay, SMMC-7721 cells were stably transfected with TMEM16A shRNA (sc-76686-SH; Santa Cruz Biotechnology, Dallas, TX, 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 tumorigenicity assay, SMMC-7721 cells were stably transfected with TMEM16A shRNA (sc-76686-SH; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Control, 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 tumorigenicity assay, SMMC-7721 cells were stably transfected with TMEM16A shRNA (sc-76686-SH; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Migration, Knockdown, Transfection, MTT Assay, Control, 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 tumorigenicity assay, SMMC-7721 cells were stably transfected with TMEM16A shRNA (sc-76686-SH; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Transfection, Flow Cytometry, Staining, Control, 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 tumorigenicity assay, SMMC-7721 cells were stably transfected with TMEM16A shRNA (sc-76686-SH; Santa Cruz Biotechnology, Dallas, TX, 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 tumorigenicity assay, SMMC-7721 cells were stably transfected with TMEM16A shRNA (sc-76686-SH; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Knockdown, In Vivo, shRNA, Transfection, Negative Control

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: Frontiers in Immunology

Article Title: Age-Related Differences in Structure and Function of Nasal Epithelial Cultures From Healthy Children and Elderly People

doi: 10.3389/fimmu.2022.822437

Figure Lengend Snippet: Age-related differences in calcium-activated chloride secretion in nasal epithelial cultures from healthy children compared to elderly people. (A, B) Representative original recordings of transepithelial Ussing chamber measurements in primary nasal epithelial cultures from children and elderly people. (C–G) Summary of individual effects of basal I sc (C) , amiloride-sensitive I sc (D) , amiloride-insensitive I sc (E) , cAMP-activated I sc (F) , CFTR inhibitor 172-sensitive I sc (G) , and UTP-activated I sc (H) ( n = 17 and 14 individuals per group, data represent mean values of 2–3 filters per individual). (I, J) Transcript levels of CFTR (I) and TMEM16A (J) ( n = 16 and 12 individuals per group). * p < 0.05 compared to children. Data are shown as mean ± S.E.M. Statistical analysis was performed with unpaired two-tailed t -test in (D–G) , and with two-tailed Mann–Whitney test in (C, H–J) .

Article Snippet: The primary antibodies used were rat monoclonal anti-α-tubulin (mAb1864, Millipore, Burlington, MA, USA), mouse monoclonal anti-MUC5AC (sc-59951, Santa Cruz, Dallas, TX, USA), and rabbit polyclonal anti-KRT5 (SAB1410739, Sigma, St. Louis, MO, USA) at dilution of 1:200 for 1 h. For TMEM16A localization, rabbit polyclonal anti-TMEM16A antibody (HPA032148, Atlas Antibodies, Stockholm, Sweden) was used at a dilution of 1:50 overnight at 4°C.

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Age-Related Differences in Structure and Function of Nasal Epithelial Cultures From Healthy Children and Elderly People

doi: 10.3389/fimmu.2022.822437

Figure Lengend Snippet: Age-related differences in calcium-activated chloride secretion in nasal epithelial cultures are mediated by TMEM16A. (A–E) Representative original recordings and summary data of transepithelial Ussing chamber measurements in primary nasal epithelial cultures from healthy children (A, B, E) and elderly people (C–E) showing the effect of UTP-induced I sc in the absence (A, C, E) and presence (B, D, E) of the TMEM16A inhibitor Ani9 ( n = 4 and 9 individuals per group, data represent mean values of 2–3 filters per individual). * p < 0.05 and ** p < 0.01 compared to Ani9- group. Data are shown as mean ± S.E.M. Statistical analysis was performed with paired two-tailed t test in (E) .

Article Snippet: The primary antibodies used were rat monoclonal anti-α-tubulin (mAb1864, Millipore, Burlington, MA, USA), mouse monoclonal anti-MUC5AC (sc-59951, Santa Cruz, Dallas, TX, USA), and rabbit polyclonal anti-KRT5 (SAB1410739, Sigma, St. Louis, MO, USA) at dilution of 1:200 for 1 h. For TMEM16A localization, rabbit polyclonal anti-TMEM16A antibody (HPA032148, Atlas Antibodies, Stockholm, Sweden) was used at a dilution of 1:50 overnight at 4°C.

Techniques: Two Tailed Test

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium

doi: 10.1152/ajpgi.00236.2015

Figure Lengend Snippet: Gene name, symbol, and Life Technologies assay ID for custom TaqMan mini-array

Article Snippet: The threshold cycle (C t ) of a gene of interest was subtracted from the geometric mean C t of three housekeeping genes to yield ΔC t . The relative mRNA expression of Cftr KO relative to WT enteroids was calculated using the ΔΔC t method ( 40 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Gene Name Symbol Assay ID Glucuronidase, β * Gusb Mm00446953_m1 Hypoxanthine guanine phosphoribosyl transferase 1 * Hprt1 Mm00446968_m1 Mitochondrial ribosomal protein L19 * Mrpl19 Mm00452754_m1 Sodium/potassium ATPase-α 1 (Na + -K + -ATPase) Atp1a1 Mm00523255_m1 Carbonic anhydrase II ( CA II ) Car2 Mm00501572_m1 Carbonic anhydrase IX ( CA IX ) Car9 Mm00519870_m1 Sodium-potassium-2 chloride cotransporter 1 ( Nkcc1 ) Slc12a2 Mm00436554_m1 Downregulated in adenoma ( Dra ) Slc26a3 Mm00445313_m1 Putative anion transporter 1 ( Pat-1 ) Slc26a6 Mm00506742_m1 Anion exchanger 2 ( Ae2 ) Slc4a2 Mm00436617_m1 Sodium-bicarbonate cotransporter e1 (Nbce1) Slc4a4 Mm01347935_m1 Sodium/hydrogen exchanger 1 ( Nhe1 ) Slc9a1 Mm00444270_m1 Sodium/hydrogen exchanger 2 ( Nhe2 ) Slc9a2 Mm01237129_m1 Sodium/hydrogen exchanger 3 ( Nhe3 ) Slc9a3 Mm01352473_m1 Anoctamin 1 ( Ano1 ) Ano1 Mm00724407_m1 Open in a separate window * Housekeeping gene.

Techniques:

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium

doi: 10.1152/ajpgi.00236.2015

Figure Lengend Snippet: Changes in expression of pHi regulators in Cftr KO crypt enteroids. A: quantitative real-time PCR of mRNA from 8 sex-matched WT and Cftr KO littermate pairs. Genes represented are the Na+-K+-ATPase, carbonic anhydrases (CA II and CA IX), Na+-K+-2Cl− cotransporter (Nkcc1, Slc12Aa2), downregulated in adenoma (Dra, Slc26a3), putative anion transporter-1 (Pat-1, Slc26a6), anion exchanger 2 (Ae2, Slc4a2), electrogenic NaHCO3 cotransporter 1 (Nbce1, Slc4a4), Na+/H+ exchangers 1, 2, and 3 (Nhe1-3, Slc9a1-3), and the anion channel anoctamin 1 (Ano1, Tmem16a). *Significantly different from 0 (by 1-sample t-test). B: representative immunoblot for Ae2 and β-actin of protein lysate from enteroids cultured from a sex-matched WT and Cftr KO littermate pair and a sex-matched Ae2 KO mouse. C: cumulative data for Ae2 immunoblot densitometry of enteroids from sex-matched WT and Cftr KO littermate pairs. Values are means ± SE (n = 11). *Significantly different from WT (by unpaired t-test). D: cumulative data for early growth response-1 (Egr-1) immunoblot densitometry of enteroids from sex-matched WT and Cftr KO littermate pairs. Values are means ± SE (n = 8). *Significantly different from WT (by unpaired t-test).

Article Snippet: The threshold cycle (C t ) of a gene of interest was subtracted from the geometric mean C t of three housekeeping genes to yield ΔC t . The relative mRNA expression of Cftr KO relative to WT enteroids was calculated using the ΔΔC t method ( 40 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Gene Name Symbol Assay ID Glucuronidase, β * Gusb Mm00446953_m1 Hypoxanthine guanine phosphoribosyl transferase 1 * Hprt1 Mm00446968_m1 Mitochondrial ribosomal protein L19 * Mrpl19 Mm00452754_m1 Sodium/potassium ATPase-α 1 (Na + -K + -ATPase) Atp1a1 Mm00523255_m1 Carbonic anhydrase II ( CA II ) Car2 Mm00501572_m1 Carbonic anhydrase IX ( CA IX ) Car9 Mm00519870_m1 Sodium-potassium-2 chloride cotransporter 1 ( Nkcc1 ) Slc12a2 Mm00436554_m1 Downregulated in adenoma ( Dra ) Slc26a3 Mm00445313_m1 Putative anion transporter 1 ( Pat-1 ) Slc26a6 Mm00506742_m1 Anion exchanger 2 ( Ae2 ) Slc4a2 Mm00436617_m1 Sodium-bicarbonate cotransporter e1 (Nbce1) Slc4a4 Mm01347935_m1 Sodium/hydrogen exchanger 1 ( Nhe1 ) Slc9a1 Mm00444270_m1 Sodium/hydrogen exchanger 2 ( Nhe2 ) Slc9a2 Mm01237129_m1 Sodium/hydrogen exchanger 3 ( Nhe3 ) Slc9a3 Mm01352473_m1 Anoctamin 1 ( Ano1 ) Ano1 Mm00724407_m1 Open in a separate window * Housekeeping gene.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture