Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Blocking Assay, Knockdown, Control

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Produced, Concentration Assay

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Functional Assay, Control

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Blocking Assay, Control

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Imaging, Labeling

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

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

Journal: The Journal of general physiology

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

doi: 10.1085/jgp.202213217

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

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

Techniques: Membrane, Activation Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 1. Isolation of ALC1 from 1q21 amplicon. (A) Amplification of different regions along chromosome 1q in 60 HCC cases was detected by FISH. The number of cases amplified is indicated above each bar. (B) FISH analysis showed that the amplified microdissected DNA probe was specifically hybridized to normal chromosome 1q21 (red signals). This microdissected DNA was used for cDNA selection from an HCC case with 1q21 amplification. (C) ALC1 was mapped to 1q21 by FISH with a BAC clone containing ALC1 (indicated by arrows). (D) A representative exam- ple of ALC1 gene amplification detected in H-4 cells by FISH with the BAC probe (red signals). A BAC probe from 1p32 (green signals) was used as a control. (E) ALC1 was cloned to pEGFP vector. The exogenously expressed ALC1-EGFP fusion protein (green color) was sublocalized in the nucleus. (F) Compared to CHD1, the predicted protein structure of ALC1 also has SNF2_N and HELICc domains.

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Isolation, Amplification, Selection, Control, Clone Assay, Plasmid Preparation

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 2. Overexpression of ALC1 in primary HCCs and HCC cell lines. (A) A representative example of ALC1 amplification in HCC TMA detected by FISH with the BAC clone contain- ing ALC1. (B) A 98-kD protein was detected by anti-ALC1 antibody. (C) Positive nuclear staining of ALC1 was frequently detected in primary HCC (left) but not in its matched adjacent nontumor tissue specimen (right) by IHC. (D) The IHC result in TMA was verified on a larger tissue section containing HCC tissue (upper part) and surrounding nontumor liver tissue (lower part). RNA expression of ALC1 was tested in (E) primary HCC cases and(F) HCC cell lines by north- ern blot analysis. In primary HCC, ALC1 expression was compared be- tween tumors (T) and their matched nontumor liver tissues (N).

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Over Expression, Staining, RNA Expression, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 3. Oncogenic ability of ALC1. (A) Expression of ALC1 in ALC1- transfected LO2 and QGY-7703 cells detected by northern blot hybrid- ization. Blank vector–transfected cells were used as controls. (B) Rates of colony formation in soft agar detected in ALC1-transfected and blank vector–transfected LO2 and QGY-7703 cells (**P 0.05). (C,D) Rep- resentative examples of tumors formed in nude mice following injection of ALC1-expressing LO2 cells (left) and QGY-7703 cells (right). ALC1- expressing cells and mock cells were injected into the right and left dorsal flanks, respectively. (E) Flow cytometry histogram showing that overex- pression of ALC1 in ALC1-expressing QGY-7703 cells could promote G1/S phase transition compared to vector-transfected QGY-7703 cells.

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Expressing, Transfection, Northern Blot, Plasmid Preparation, Injection, Flow Cytometry, Sublimation

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 4. Silencing ALC1 expression by siRNA. (A) Two siRNAs (ALC1-si1 and ALC1-si2) could efficiently reduce the expression of ALC1 in H2-M cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control. (B) Colony formation ability in soft agar was decreased significantly in siRNA-treated H2-M cells (**P 0.05). (C) Flow cytometry analysis showed that ALC1-si1 could inhibit the cell cycle at the G1/S checkpoint. The percentage of cells in the S phase was decreased from 35% to 23.7%. (D) Western blot analyses indicated that p53 and p21Waf1/Cip1 were down-regulated, whereas cyclin E and Cdk2 were up-regulated in ALC1-transfected QGY-7703 cells in comparison with vector-transfected QGY-7703 cells. -Actin was used as a loading control. (E) Western blot results were quantified by densitometry, and data are presented as mean standard error (n 3). Fold values were first normalized with actin and then compared with vector-transfected QGY-7703 cells.

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Expressing, Control, Flow Cytometry, Western Blot, Transfection, Comparison, Plasmid Preparation

Journal: Hepatology (Baltimore, Md.)

Article Title: Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma.

doi: 10.1002/hep.22072

Figure Lengend Snippet: Fig. 5. The inhibition role of ALC1 in apoptosis. (A) Representative figures of TUNEL staining images. After cells were treated with STS for 4 hours, more apoptotic cells (bright white) were detected in vector- transfected QGY-7703 cells in comparison with ALC1-transfected 7703 cells. (B) Detection of the apoptotic index between ALC1-transfected and vector-transfected QGY-7703 cells before and after STS treatment (**P 0.05). The data showed that ALC1-transfected QGY-7703 cells could resist STS-induced apoptosis in comparison with QGY-7703 only. (C) Expressions of caspase 3 and Bax were compared between ALC1- transfected and vector-transfected QGY-7703 cells before and after STS treatment by western blot analyses. -Actin was used as a loading control. (D) Protein levels of caspase 3 and Bax were quantified by densitometry, and data are shown as mean standard error (n 3).

Article Snippet: TMA sections were deparaffinized and incubated with polyclonal anti-ALC1 antibody (Boster Biotechnology Co., Ltd., Wuhan, China) in a dilution of 1:100 at 4°C overnight.

Techniques: Inhibition, TUNEL Assay, Staining, Plasmid Preparation, Transfection, Comparison, Western Blot, Control