Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2–Nrf2–CEBPB Transcriptional Axis in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms25179612

Figure Lengend Snippet: Functional expression of LRRC8A in M 2 -MACs. ( A , B ) Simultaneous measurement of changes in membrane potential ( A ) and [Ca 2+ ] i ( B ) following the application of the LRRC8/ANO1 inhibitor, endovion (EDV, 1 μM), using bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC 4 (3)] and Fura 2-acetoxymethyl ester (Fura 2-AM), respectively. The relative time course of changes in fluorescence intensities (1.0 at time 0 s) from an M 2 -MAC is shown. ( C , D ) Summarized results of EDV (1 μM)-induced depolarization (at 1 min) ( C ) and hyperpolarization (at 10 min) ( D ) responses. ( E , F ) Summarized results of EDV (1 μM)-induced changes in [Ca 2+ ] i at 1 min ( E ) and 10 min ( F ). ( G , H ) Measurement of changes in membrane potential following the application of the selective ANO1 inhibitor, ANO1-IN-1 (1 μM), using DiBAC 4 (3). The relative time course of changes in fluorescence intensities (1.0 at time 0 s) from an M 2 -MAC is shown ( G ). Summarized results of ANO1-IN-1-induced responses ( H ). Numbers used for experiments are shown in parentheses. **: p < 0.01 vs. the vehicle control.

Article Snippet: EDV (HY-105917), ANO1-IN-1 (HY-16320), NK252 (HY-19734), SU056 (HY-50231), and SKA121 (HY-107414) were from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Functional Assay, Expressing, Membrane, Fluorescence, Control

Journal: American journal of physiology. Cell physiology

Article Title: Mouse organoid culture is a suitable model to study esophageal ion transport mechanisms.

doi: 10.1152/ajpcell.00295.2021

Figure Lengend Snippet: Figure 3. Expression of ion transporters in esophageal organoids (EOs). A: mature EOs were collected 9 days after plating, and RNA was prepared from the organoids. Gene expression of ion transporters was investigated with traditional RT-PCR analysis. B: immunostaining of EOs for Slc9a1 (first line), Slc9a2 (second line), Slc26a3 (third line), Slc4a4 (fourth line), and ANO1 (fifth line). The scale bar represents 100 mm (main photo) and 50 mm (inset photo), respectively. C: costaining of Slc26a6 (red) and cftr (green). The scale bar represents 50 mm (upper line), 25 mm (middle line), and 10 mm (bottom line), for both mice strains. CFTR, cystic fibrosis transmembrane conductance regulator.

Article Snippet: List of primary and secondary antibodies used in the study Primary Antibodies Company Cat. No. Dilution Slc9a1 Alomone ANX-010 1:100 Slc9a2 Alomone ANX-002 1:100 Slc26a3 Invitrogen PA5-68530 1:600 Slc26a6 Santa Cruz Biotechnology sc-515230 1:200 Slc4a4 Abcam ab187511 1:100 CFTR Alomone ACL-006 1:200 ANO1 Alomone ACL-011 1:200 CFTR, cystic fibrosis transmembrane conductance regulator.

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Immunostaining

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: 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: 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, 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 knockdown of TMEM16A, the TMEM16A siRNA and negative siRNA (50 nM) were synthesized and purified by OriGene (OriGene, Rockville, MD, 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 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, 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 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: Knockdown, In Vivo, shRNA, Transfection, Negative Control

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: KN-93 reduces the surface expression and activity of ANO1 in U251 cells. ( A ) U251 cells treated with DMSO or KN-93 were imaged using antibodies against ANO1 and WGA647 (WGA), a plasma membrane marker. Scale bar, 20 μm. ( B ) The Pearson’s correlation coefficient for ANO1 with KN-93 was significantly less than the value obtained for ANO1 with DMSO in U251 cells. ( C ) Cell surface biotinylation results from membrane protein fractions from U251 cells treated with DMSO or KN-93. ( D ) The summary bar graph showing data obtained from three independent experiments as in ( C ). ( E ) Averaged traces of whole-cell currents of U251 cells treated with DMSO or T16Ainh-A01, an ANO1 inhibitor. ( F ) The summary bar graph shows the inhibitory effect of KN93 or T16Ainh-A01 on ANO1 current amplitude at ±100 mV. ( G ) Averaged traces of normalized T16Ainh-A01-sensitive currents of U251 cells treated with DMSO or KN-93. ( H ) The bar graph shows normalized T16Ainh-A01-sensitive current densities ( G ) at + 100 mV. Number on each bar indicates n for each condition. All values are mean ± s.e.m. P -values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001. n.s means not significant.

Article Snippet: To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene (Rockville, Maryland, USA, TL300993), which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D).

Techniques: Expressing, Activity Assay, Clinical Proteomics, Membrane, Marker

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: CaMKIIβ specifically increases the surface expression and activity of ANO1 in U251 cells. ( A ) The mRNA expression of CaMKII isoforms in U251 cells were detected with specific primers for CaMKIIα, β, γ and δ. ( B ) Normalized expression of CaMKII isoforms as compared to that of GAPDH in U251 cells. ( C ) Effect of CaMKII isoform overexpression on ANO1-mediated whole cell chloride currents in U251 cells. ( D ) The summary bar graph showing ANO1-mediated current density at ± 100 mV. ( E ) Cell surface biotinylation results from membrane protein fractions from U251 cells transfected with mCh-CaMKIIα and mCh-CaMKIIβ. ( F ) The summary bar graph showing data obtained from at least three independent experiments as in ( E ). Number on each bar indicates n for each condition. All values are mean ± s.e.m. P -values were obtained with Student’s t-test. ** p < 0.01 and *** p < 0.001. n.s means not significant.

Article Snippet: To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene (Rockville, Maryland, USA, TL300993), which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D).

Techniques: Expressing, Activity Assay, Over Expression, Membrane, Transfection

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: CaMKIIβ knockdown reduces the surface expression of ANO1 in U251 cells. ( A,B ) Validation of the silencing efficiency of the siRNA against CaMKIIβ using qPCR and Western blotting. ( C ) U251 cells transfected with Sc shRNA or CaMKIIβ siRNA were imaged using antibodies against ANO1 and WGA. Nuclei were stained using DAPI staining solution. Scale bar, 20 μm. ( D ) Cell surface biotinylation results from U251 cells transfected with Sc shRNA or CaMKIIβ siRNA. ( E ) The summary bar graph shows the summary of ( D ), data obtained from three independent experiments. ( F ) Averaged traces of whole-cell currents of U251 cells transfected with Sc shRNA, CaMKIIβ siRNA, or T16Ainh-A01 (A01). ( G ) The summary bar graph shows the inhibitory effect of CaMKIIβ siRNA on ANO1–mediated current amplitude at ±100 mV. The bar graph shows normalized A01-sensitive current densities at + 100 mV. Number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with Student’s t-test. * p < 0.05 and ** p < 0.01.

Article Snippet: To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene (Rockville, Maryland, USA, TL300993), which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D).

Techniques: Knockdown, Expressing, Biomarker Discovery, Western Blot, Transfection, shRNA, Staining

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: Gene silencing of ANO1 or CaMKIIβ leads to the attenuation of migration and invasion in U251 cells. ( A,B ) Validation of the silencing efficiency of the shRNA against ANO1 using qPCR and Western blotting. ( C ) Cell invasion assay of U251 cells infected with Lenti-ANO1 shRNA and transfected with CaMKIIβ siRNA. ( D ) The averaged bar graph shows the summary of ( C ), data obtained from three independent experiments. ( E ) Representative photographic images of the migration of U251 cells infected with Lenti-ANO1 shRNA and transfected with CaMKIIβ siRNA and the cells with Lenti-Sc shRNA and Sc siRNA control. Scale bar, 100 μm. ( F ) The averaged bar graph shows the summary of ( E ), data obtained from three independent experiments. All values are mean ± s.e.m. p -values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene (Rockville, Maryland, USA, TL300993), which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D).

Techniques: Migration, Biomarker Discovery, shRNA, Western Blot, Invasion Assay, Infection, Transfection, Control

Journal: Cells

Article Title: Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

doi: 10.3390/cells9051079

Figure Lengend Snippet: Inhibition of CaMKIIβ and/or ANO1 decreased migration and invasion of U87MG cells. ( A,B ) Validation of the silencing efficiency of the Lenti-ANO1shRNA and the CaMKIIβ siRNA with qPCR and Western blot in U87MG cells. ( C ) Cell invasion assay of U87MG cells infected with Lenti-ANO1shRNA and transfected with CaMKIIβ siRNA. ( D ) The averaged bar graph shows the summary of (C), data obtained from three independent experiments. ( E ) Representative photographic images of the migration of U87MG cells infected with Lenti-ANO1shRNA and transfected with CaMKIIβ siRNA as compared to the cells with Lenti-Sc shRNA and Sc siRNA control. Scale bar, 100 μm. ( F ) The averaged bar graph shows the summary of ( E ), data obtained from three independent experiments. All values are mean ± s.e.m. p -values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene (Rockville, Maryland, USA, TL300993), which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D).

Techniques: Inhibition, Migration, Biomarker Discovery, Western Blot, Invasion Assay, Infection, Transfection, shRNA, Control

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: Biomedicines

Article Title: Arctigenin Attenuates Vascular Inflammation Induced by High Salt through TMEM16A/ESM1/VCAM-1 Pathway

doi: 10.3390/biomedicines10112760

Figure Lengend Snippet: ( A ) Western blot analysis indicated increased expression of TMEM16A in the HS group compared with NS group. ( B ) Relative level of TMEM16A protein expression. Data are expressed as mean ± SD, * p < 0.05, t -test ( n = 3). ( C – E ) Representative images of TMEM16A (green) immunofluorescence in thoracic aortas in the NS group, HS group, and HS+ARC100 groups ( n = 4). Scale bar: 50 µm; blue represents nucleus. ( C ) NS group; ( D ) HS group; ( E ) HS+ARC100 group. ( F ) Mean fluorescence was quantified using one-way ANOVA and p -value adjusted for multiple comparisons with Sidak multiple-comparison test ( n = 4), **** p < 0.0001. NS: normal salt group, HS: high-salt group, HS+ARC: HS+ arctigenin (100 mg/kg) group.

Article Snippet: Small interfering RNA targeting the TMEM16A gene (siTEME16A) was purchased from OriGene (CAT No: SR411408, OriGene China, Wuxi, China).

Techniques: Western Blot, Expressing, Immunofluorescence, Fluorescence, Comparison

Journal: Biomedicines

Article Title: Arctigenin Attenuates Vascular Inflammation Induced by High Salt through TMEM16A/ESM1/VCAM-1 Pathway

doi: 10.3390/biomedicines10112760

Figure Lengend Snippet: ( A ) Western blotting results showed discernible reduction in TMEM16A proteins in VSMCs after treating with 50, 100, and 200 μM arctigenin for 24 h, mean protein was quantified using one-way ANOVA and p -value adjusted for multiple comparisons with Sidak multiple-comparison test ( n = 3), **** p < 0.0001; ( B ) 30 μM siTMEM16A knockdown TMEM16A protein for 24 h in the TMEM16A knockdown cells compared to the HS group. Data are expressed as mean ± SD, ** p < 0.01 t -test ( n = 3); ( C ) The Western blotting results showed that incubating aortic VSMCs with 10 μM T16Ainh-A01, 100 μM arctigenin and 30 μM siRNA TMEM16A led to a decrease in TMEM16A, VCAM-1 and ICAM-1. Data are expressed as mean ± SD, and relative protein expression was quantified using one-way ANOVA and p -value adjusted for multiple comparisons with Sidak multiple-comparison test ( n = 3), **** p < 0.0001. ( D ) Microscopic images showed immunofluorescence staining of VCAM-1 and ICAM-1 (green) in thoracic aortas ( n = 4); blue represents nucleus. Mean fluorescence was quantified using one-way ANOVA and p -value adjusted for multiple comparisons with Sidak multiple-comparison test ( n = 4), *** p < 0.001. NS: normal salt group, HS: high-salt group, T16: HS+ T16Ainh-A01 group, ARC: HS+ arctigenin (100 mg/kg) group, siTMEM16A: HS+siRNA TMEM16A group. siEMS1: HS+ siRNA ESM1 group.

Article Snippet: Small interfering RNA targeting the TMEM16A gene (siTEME16A) was purchased from OriGene (CAT No: SR411408, OriGene China, Wuxi, China).

Techniques: Western Blot, Comparison, Knockdown, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Biomedicines

Article Title: Arctigenin Attenuates Vascular Inflammation Induced by High Salt through TMEM16A/ESM1/VCAM-1 Pathway

doi: 10.3390/biomedicines10112760

Figure Lengend Snippet: ( A ) The Western blotting results showed that incubating aortic VSMCs with 10 μM T16Ainh-A01, 100 μM arctigenin, and 30 μM siRNA TMEM16A led to a decrease in ESM1. Data are expressed as mean ± SD, and relative protein expression was quantified using one-way ANOVA and p -value adjusted for multiple comparisons with Sidak multiple-comparison test ( n = 3), **** p < 0.0001. ( B ) Microscopic images showed immunofluorescence staining of ESM1 (green) in thoracic aortas ( n = 4); blue represents nucleus. Mean fluorescence was quantified using one-way ANOVA and p -value adjusted for multiple comparisons with Sidak multiple-comparison test ( n = 4), *** p < 0.001, **** p < 0.0001. ( C ) 30uM siESM1 knockdown EMS1 protein for 48 h in the ESM1 knockdown cells compared to the HS group. Data are expressed as mean ± SD, * p < 0.05, t -test ( n = 3). ( D ) ESM1, VCAM-1, and ICAM-1 protein expression dramatically declined after silencing ESM1 in VSMCs. Data are expressed as mean ± SD, and relative protein expression was quantified using one-way ANOVA and p -value adjusted for multiple comparison with Sidak multiple-comparison test ( n = 3), * p < 0.05, ** p < 0.01, **** p < 0.0001. ( E ) Aortic VSMCs were cultured with NS, HS, ARC, siTMEM16A siESM1 for 24 h and T16 for 30 min, and the levels of CXCL16 in the culture media were quantified using the enzyme-linked immunosorbent assay (ELISA). Data are expressed as mean ± SD, and relative CXCL16 expression was quantified using one-way ANOVA and p -value adjusted for multiple comparisons with Sidak multiple-comparison test ( n = 3), **** p < 0.0001 ( n = 3). NS: normal salt group, HS: high-salt group, T16: HS+ T16Ainh-A01 group, ARC: HS+ arctigenin (100 mg/kg) group, siTMEM16A: HS+siRNA TMEM16A group. siEMS1: HS+ siRNA ESM1 group.

Article Snippet: Small interfering RNA targeting the TMEM16A gene (siTEME16A) was purchased from OriGene (CAT No: SR411408, OriGene China, Wuxi, China).

Techniques: Western Blot, Expressing, Comparison, Immunofluorescence, Staining, Fluorescence, Knockdown, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cancer science

Article Title: Transcriptional repression of human epidermal growth factor receptor 2 by ClC-3 Cl - /H + transporter inhibition in human breast cancer cells.

doi: 10.1111/cas.13715

Figure Lengend Snippet: FIGURE 2 Effects of siRNA‐mediated inhibition of ClC‐3, ClC‐7, and chloride intracellular channel protein 1 (CLIC)1 on expression levels of human epidermal growth factor receptor 2 (HER2) transcripts in MDA‐MB‐453 cells. A, Real‐time PCR assay for HER2 in control siRNA (si‐cont)‐, ANO1 siRNA (si‐ANO1)‐, ClC‐3 siRNA (si‐ ClC‐3)‐, ClC‐7 siRNA (si‐ClC‐7)‐, and CLIC1 siRNA (si‐CLIC1)‐ transfected MDA‐MB‐453 cells for 72 h. Expression levels were expressed as a ratio to β‐actin (ACTB). B‐D, Protein lysates of si‐ cont‐, si‐ANO1‐, si‐ClC‐3‐, si‐ClC‐7‐, and si‐CLIC1‐transfected MDA‐ MB‐453 cells were probed by immunoblotting with anti‐HER2 and anti‐ACTB antibodies on the same filter (B, C). Summarized results were obtained as described in Section 2.5 from HER2 and ACTB band signals (D). After compensation, the HER2 signal in the si‐cont group was expressed as 1.0 (n = 4 for each). Results are expressed as means ± SEM. **P < .01 vs si‐cont. ANO, anoctamine

Article Snippet: Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) was used in all siRNA transfection procedures.19 Commercially available siRNA oligonucleotides against ClC‐3, ClC‐4, ClC‐5, ClC‐7, CLIC1, ANO1, and control siRNA were purchased from Santa Cruz Biotech- nology (Santa Cruz, CA, USA) or Thermo Fisher Scientific.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Control, Transfection, Western Blot

Journal: Cancer science

Article Title: Transcriptional repression of human epidermal growth factor receptor 2 by ClC-3 Cl - /H + transporter inhibition in human breast cancer cells.

doi: 10.1111/cas.13715

Figure Lengend Snippet: FIGURE 9 Effects of siRNA‐mediated inhibition of ClC‐3 and ANO1 on expression levels of human epidermal growth factor receptor 2 (HER2)‐related transcriptional factor transcripts in MDA‐MB‐453 and YMB‐1 cells, respectively. Real‐time PCR assay for AP‐2α (A, H), AP‐2β (B, I), AP‐2γ (C, J), STAT3 (D, K), EBP1 (E, L), MBP‐1 (F, M), and HDAC6 (G, N) in control siRNA (si‐cont) and ClC‐3 siRNA (si‐ClC‐3)‐ transfected MDA‐MB‐453 cells for 72 h (A‐G) and ANO1 siRNA (si‐ANO1)‐transfected YMB‐1 cells for 72 h (H‐N), respectively. Expression levels were expressed as a ratio to ACTB (n = 4 for each). Results are expressed as means ± SEM. ANO, anoctamine; AP, activator protein; EBP, ErbB3‐binding protein; HDAC, histone deacetylase; MBP‐1, major basic protein 1; STAT, signal transducer and activator of transcription

Article Snippet: Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) was used in all siRNA transfection procedures.19 Commercially available siRNA oligonucleotides against ClC‐3, ClC‐4, ClC‐5, ClC‐7, CLIC1, ANO1, and control siRNA were purchased from Santa Cruz Biotech- nology (Santa Cruz, CA, USA) or Thermo Fisher Scientific.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Control, Transfection, Binding Assay, Histone Deacetylase Assay