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: 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: 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: American Journal of Physiology - Cell Physiology

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

doi: 10.1152/ajpcell.00328.2017

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

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

Techniques:

Journal: American Journal of Physiology - Cell Physiology

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

doi: 10.1152/ajpcell.00328.2017

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

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

Techniques:

Journal: American Journal of Physiology - Cell Physiology

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

doi: 10.1152/ajpcell.00328.2017

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

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

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Cell Physiology

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

doi: 10.1152/ajpcell.00328.2017

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

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

Techniques: Immunohistochemistry, Expressing, Staining

Journal: Cancer Research

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

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

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

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

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

Journal: Cancer Research

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

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

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

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

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

Journal: Cancer Research

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

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

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

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

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

Journal: Cancer Research

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

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

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

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

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

Journal: Cancer Research

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

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

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

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

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

Journal: Cancer Research

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

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

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

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

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

Journal: Cancer Research

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

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

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

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

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

Journal: Cancer Research

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

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

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

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

Techniques: Generated

Journal: Therapeutics and Clinical Risk Management

Article Title: Small bowel gastrointestinal stromal tumor: a retrospective study of 32 cases at a single center and review of the literature

doi: 10.2147/TCRM.S167248

Figure Lengend Snippet: Ileal GISTs and immunohistochemistry. Notes: A 55-year-old male with an ileal GISTs presented with melena. ( A1 ) Axial plain CT image does not reveal the primary tumor due to polymorphous intestine. ( A2 ) Axial contrast-enhanced CT image shows a 1.3×1.8 cm 2 tumor (arrow) with marked enhancement in the arterial phase. ( A3 ) DBE shows the intraluminal component of the tumor with engorged vessels. ( A4 ) Gross appearance of the operative specimen shows the extraluminal component of the tumor (arrow). ( B1 ) Histopathology shows the tumor cells are composed of spindle cells with a low mitotic count (≤5 mitoses/50 HPFs; HE staining). Tumor cells are strongly positive for CD117 ( B2 ), CD34 ( B3 ), and DOG1 ( B4 ). Tumor cells are negative for SMA ( C1 ), which is positive in vascular wall. S-100 ( C2 ) and Desmin ( C3 ) are negative in tumor cells. Ki-67 ( C4 ) is positive in some of the tumor nuclei (global index 2%). (Original magnification: B1 ×100; B2 – C4 ×200). Abbreviations: GISTs, gastrointestinal stromal tumors; CT, computed tomography; DBE, double-balloon enteroscopy; HPFs, high-power fields; HE, hematoxylin and eosin; SMA, smooth muscle actin.

Article Snippet: The routine diagnostic immunohistochemistry technique used a panel of seven primary antibodies purchased from Boster Biological Technology (Wuhan, China), which included a mouse anti-CD117 monoclonal antibody used at 1:100 dilution, a rabbit anti-CD34 polyclonal antibody used at 1:200 dilution, a mouse anti-DOG1 monoclonal antibody used at 1:200 dilution, a mouse anti-SMA monoclonal antibody used at 1:150 dilution, a mouse anti-S100 monoclonal antibody used at 1:50 dilution, a mouse anti-desmin monoclonal antibody used at 1:100 dilution, and a rabbit anti-Ki-67 monoclonal antibody used at 1:100 dilution.

Techniques: Immunohistochemistry, Histopathology, Staining, Computed Tomography

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Blocking Assay, Knockdown, Control

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Produced, Concentration Assay

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Functional Assay, Control

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Blocking Assay, Control

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Imaging, Labeling

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Membrane, Activation Assay