Journal: Frontiers in pharmacology

Article Title: Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFβ1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

doi: 10.3389/fphar.2020.00165

Figure Lengend Snippet: FIGURE 1 | Large-conductance and Ca2+-activated K+ (BK) channels are expressed and functional in activated hepatic stellate cells (HSCs). (A,B) Representative RT-qPCR (A) and western blots (B) showing the expression of ACTA2 and the BK channels alpha subunit KCNMA1 in activated human LX2 cells treated with transforming growth factor beta 1 (TGFβ1) and spontaneously activated primary rat HSCs in vitro. (C) Representative immunofluorescence images of KCNMA1 and ACTA2 in activated primary rat HSCs (Scale bars, 25 µm). (D,E) Representative whole-cell K+ current traces and the normalized current recorded from activated HSCs before and after treatment with rottlerin (Rot) and paxilline (Pax), as indicated at 1 µM internal Ca2+. The whole-cell currents were elicited by 1 s voltage ramps from −100 to 80 mV. Rottlerin (1 µM) and paxilline (10 µM) were freshly prepared from stock solutions and the final DMSO concentration was 0.1% (*p < 0.05 compared with the vehicle group, n = 3).

Article Snippet: Antibodies against SMAD3, p-SMAD3, JAK2, p-JAK2, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, United States), the anti-α-SMA (ACTA2) antibody was from Novus Biology (Centennial, CO, United States), and the anti-BK-Slo1 was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Functional Assay, Quantitative RT-PCR, Western Blot, Expressing, In Vitro, Concentration Assay

Journal: Frontiers in pharmacology

Article Title: Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFβ1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

doi: 10.3389/fphar.2020.00165

Figure Lengend Snippet: FIGURE 2 | Overexpression of the BK channel alpha subunit (KCNMA1) inhibits hepatic stellate cell (HSC) migration and fibrosis-related gene expression. (A) Validation of KCNMA1 overexpression by qPCR (left) and western blotting (right). (B) Representative images of cells (left) and average number of migrated cells (right) showing the inhibitory effects of transient KCNMA1 overexpression on LX2 cell migration. (C) Normalized mRNA expression of aortic smooth muscle actin (ACTA2), collagen type I alpha 1 chain (COL1A1), COL1A2, and C-C motif chemokine ligand 2 (CCL2) in LX2 cells pretreated with TGFβ1 after transfection with KCNMA1-expressing plasmids. (D) Detection of KCNMA1 by qPCR (left) and western blotting (right) after siRNA transfection. (E) Representative images (left) and averaged number of migrated cells (right) showing the inhibitory effects of knocking down KCNMA1 on LX2 cell migration. (F) Normalized mRNA expression levels of ACTA2, COL1A1, COL1A2, and CCL2 in LX2 cells pretreated with TGFβ1 after transfection with siRNA targeting KCNMA1. (G) Ratio of EdU-positive cells to Hoechst 33342-stained nuclei after transfection of KCNMA1-expressing plasmids (left) or KCNMA1 siRNA (right) (∗p < 0.05, ∗∗p < 0.01 compared with the vector or negative control (NC) group.

Article Snippet: Antibodies against SMAD3, p-SMAD3, JAK2, p-JAK2, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, United States), the anti-α-SMA (ACTA2) antibody was from Novus Biology (Centennial, CO, United States), and the anti-BK-Slo1 was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Over Expression, Migration, Gene Expression, Biomarker Discovery, Western Blot, Expressing, Transfection, Staining, Plasmid Preparation, Negative Control

Journal: Frontiers in pharmacology

Article Title: Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFβ1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

doi: 10.3389/fphar.2020.00165

Figure Lengend Snippet: FIGURE 3 | Upregulation of large-conductance and Ca2+-activated K+ (BK) channel activity by rottlerin inhibits hepatic stellate cell (HSC) migration and fibrosis-related gene expression in vitro. (A,B) Representative images (left) and averaged number of migrated cells (right) showing the inhibitory effects of rottlerin (Rot), a BK channel activator, on the migration of human LX2 cells (A) and activated primary rat HSCs (B) at the indicated concentrations (∗p < 0.05, ∗∗p < 0.01 compared with vehicle). (C) Relative mRNA expression levels of ACTA2, COL1A1, COL1A2, and CCL2 in human LX2 cells with the indicated treatments for 24 h. (D) Normalized cell viability of LX2 cells measured under the indicated conditions (∗∗p < 0.01 compared with vehicle, ##p < 0.01 compared with the TGFβ1-treated group).

Article Snippet: Antibodies against SMAD3, p-SMAD3, JAK2, p-JAK2, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, United States), the anti-α-SMA (ACTA2) antibody was from Novus Biology (Centennial, CO, United States), and the anti-BK-Slo1 was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Activity Assay, Migration, Gene Expression, In Vitro, Expressing

Journal: Frontiers in pharmacology

Article Title: Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFβ1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

doi: 10.3389/fphar.2020.00165

Figure Lengend Snippet: FIGURE 4 | Treatment with the large-conductance and Ca2+-activated K+ (BK) channel activator rottlerin ameliorates CCl4-induced liver fibrosis in vivo. (A) Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) from rats in the indicated groups (∗∗p < 0.01 compared with the vehicle (Veh) group, #p < 0.05 and ##p < 0.01 compared with the CCl4-treated group, n = 5). (B) Relative mRNA levels of ACTA2 in the liver tissue of rats subjected to the indicated treatments (∗∗p < 0.01 compared with the Veh group, ##p < 0.01 compared with the CCl4-treated group, n = 5). (C) Representative western blots (left) and normalized intensity (right) of the indicated ACTA2 protein levels in rats subjected to the indicated treatments (∗∗p < 0.01 compared with the Veh group, ##p < 0.01 compared with the CCl4-treated group, n = 5). (D) Representative images of liver sections stained with hematoxylin and eosin (H&E), anti-ACTA2 antibody, and Sirius Red (left) and quantification of the positive area (right), in rats subjected to the indicated treatments (∗∗p < 0.01 compared with the Veh group, ##p < 0.01 compared with the CCl4-treated group, scale bar: 50 µm, n = 5).

Article Snippet: Antibodies against SMAD3, p-SMAD3, JAK2, p-JAK2, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, United States), the anti-α-SMA (ACTA2) antibody was from Novus Biology (Centennial, CO, United States), and the anti-BK-Slo1 was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: In Vivo, Western Blot, Staining

Journal: Journal of thoracic disease

Article Title: Deletion of ACTA2 in mice promotes angiotensin II induced pathogenesis of thoracic aortic aneurysms and dissections.

doi: 10.21037/jtd.2018.07.75

Figure Lengend Snippet: Figure 2 Increased OPN expression and decreased α-SMA expression in AngII-treated ACTA2

Article Snippet: A mouse germline ACTA2 mutation was generated in a mixed C57BL/6J background in Cyagen Biosciences of Guangdong.

Techniques: Expressing

Journal: Journal of thoracic disease

Article Title: Deletion of ACTA2 in mice promotes angiotensin II induced pathogenesis of thoracic aortic aneurysms and dissections.

doi: 10.21037/jtd.2018.07.75

Figure Lengend Snippet: Figure 4 ACTA2 deficiency rendered aortic SMC susceptible to apoptosis. The expression levels of Bax and Bcl-2 were assessed by western

Article Snippet: A mouse germline ACTA2 mutation was generated in a mixed C57BL/6J background in Cyagen Biosciences of Guangdong.

Techniques: Expressing, Western Blot

Journal: The Journal of international medical research

Article Title: Physical properties of poly(N-isopropylacrylamide) hydrogel promote its effects on cardiac protection after myocardial infarction.

doi: 10.1177/030006051204000615

Figure Lengend Snippet: FIGURE 6: Neovasculature formation in sham-operated and infarct areas of myocardium 90 days after induction of myocardial infarction (MI) in rats, as identified by immunohistochemical staining for α-smooth muscle actin. (A) Sham-operated group; (B) phosphate-buffered saline (PBS)-treated MI group; (C) Gel A-treated MI group; (D) Gel B-treated MI group (scale bars, 100 µm). Immunohistochemical staining was used to quantify (E) blood vessel density per high-power field (HPF) in each group. Black arrows show neovasculature formed in sham-operated or infarcted myocardium. aP < 0.05 compared with sham-operated group; bP < 0.05 compared with MI + PBS group; one-way analysis of variance and unpaired Student’s t-test

Article Snippet: The slides were incubated overnight at 4 °C with anti-αsmooth muscle actin primary antibody (dilution 1 : 50; Boster, Wuhan, China).

Techniques: Immunohistochemical staining, Staining, Saline

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dysregulated cholesterol uptake and efflux of bone marrow-derived α-SMA + macrophages contribute to atherosclerotic plaque formation

doi: 10.1007/s00018-025-05655-3

Figure Lengend Snippet: Macrophage-specific deletion of α-SMA inhibits atherosclerotic plaque formation. A Strategy for the generation of Acta2 f/f mice. B Genotyping results for flox1 (F1), flox2 (F2), or LysM-Cre. C Acta2 mRNA levels in BMDMs from Acta2 f/f or Acta2 MKO mice. D Schematic representation of the experimental workflow. E Representative en face images of Sudan IV-stained aortas from Acta2 f/f or Acta2 MKO mice treated with AAV -PCSK9 DY followed by a 12-week HFD. The mean aortic lesion area was quantified. F – H Representative Oil Red O-stained aortic root sections F , with quantification of lesion size G–H . Scale bar = 200 μm. n = 7 or 8. ** P < 0.01 by unpaired Student’s t-test. I , J Representative immunofluorescence staining for macrophages (MOMA-2, I ) and smooth muscle cells (α-SMA, J ) in the aortic root after a 12-week HFD. Purple, MOMA-2; red, α-SMA; blue, DAPI. Black scale bar = 200 μm, white scale bar = 50 μm. n = 7. * P < 0.05 by unpaired Student’s t-test. AAV, adeno-associated virus; HFD, high-fat diet

Article Snippet: Acta2 f/f (C57BL/6J background) mice were generated by Cyagen Biosciences (Guangzhou, China).

Techniques: Staining, Immunofluorescence, Virus

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dysregulated cholesterol uptake and efflux of bone marrow-derived α-SMA + macrophages contribute to atherosclerotic plaque formation

doi: 10.1007/s00018-025-05655-3

Figure Lengend Snippet: α-SMA promotes the lipid uptake and inhibits lipid efflux in macrophages. A , B Flow cytometric analysis of binding and uptake of DiI-Ox-LDL in vector- or Acta2 hi RAW264.7 cells A , and BMDMs from Acta2 f/f or Acta2 MKO mice B . The quantification results are shown on the right. n = 3. * P < 0.05, *** P < 0.001, **** P < 0.0001 by unpaired Student’s t-test. C , D SR-A, ABCA1, ABCG1 and α-SMA protein levels in vector- and Acta2 hi RAW264.7 cells C , and BMDMs from Acta2 Flox or Acta2 MKO mice D . n = 3. * P < 0.05 by unpaired Student’s t-test. E–F Co-staining and quantification of SR-A + MOMA-2 + E or ABCA1 + MOMA-2 + F cells in aortic roots from Acta2 f/f or Acta2 MKO mice treated with AAV -PCSK9 DY followed by a 12-week HFD. n = 7. * P < 0.05 by unpaired Student’s t-test. G–H Flow cytometric analysis of binding G and uptake H of DiI-Ox-LDL in Acta2 hi RAW264.7 cells treated with an SR-A blocking antibody (20 μg/mL) or IgG control for 2 h were detected by flow cytometry. n = 3. * P < 0.05 by unpaired Student’s t-test. AAV, adeno-associated virus; HFD, high-fat diet

Article Snippet: Acta2 f/f (C57BL/6J background) mice were generated by Cyagen Biosciences (Guangzhou, China).

Techniques: Binding Assay, Plasmid Preparation, Staining, Blocking Assay, Control, Flow Cytometry, Virus

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dysregulated cholesterol uptake and efflux of bone marrow-derived α-SMA + macrophages contribute to atherosclerotic plaque formation

doi: 10.1007/s00018-025-05655-3

Figure Lengend Snippet: AKT pathway is involved in α-SMA-induced lipid accumulation. A Abca1 mRNA levels in vector or Acta2 hi RAW264.7 cells treated with 50 μg/ml Ox-LDL for 0, 6, 12, or 24 h. n = 3. * P < 0.05 by unpaired Student’s t-test. B Top 15 KEGG pathways of genes downregulated in Acta2 hi RAW264.7 cells incubated with Ox-LDL for 6 h. C Protein levels of p-AKT, AKT, p-ERK, ERK, p-STAT3, STAT3, CD36, SR-A, ABCA1 and α-SMA treated with 50 μg/ml Ox-LDL for 6 h. n = 3. * P < 0.05 by one-way ANOVA. D – F Flow cytometric analysis of binding ( D and E ) and uptake ( D and F ) of DiI-Ox-LDL in vector or Acta2 hi RAW264.7 cells incubated with or without SC79 (2 μg/mL). n = 3. * P < 0.05, ** P < 0.01, **** P < 0.0001 by one-way ANOVA. G – I Protein levels of CD36, SR-A, ABCA1 and α-SMA in Acta2 hi or vector RAW264.7 cells incubated with or without SC79 (2 μg/mL) and treated with 50 μg/ml Ox-LDL for 2 h. n = 3. * P < 0.05, ** P < 0.01, **** P < 0.0001 by one-way ANOVA

Article Snippet: Acta2 f/f (C57BL/6J background) mice were generated by Cyagen Biosciences (Guangzhou, China).

Techniques: Plasmid Preparation, Incubation, Binding Assay