Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Caveolae‐Mediated Activation of Mechanosensitive Chloride Channels in Pulmonary Veins Triggers Atrial Arrhythmogenesis

doi: 10.1161/JAHA.119.012748

Figure Lengend Snippet: Caveolar macromolecular mechanosensitive complex. Immunofluorescent analysis of colocalized expression of ClC‐3 (left), ClC‐2 (middle), and SWELL 1 (right) chloride channels with caveolae scaffolding protein Cav3 (caveolin 3) in nonstretched rat pulmonary vein distal ( PV dis ; A ) and human left atrium ( B ) myocardium. For colocalization analysis, intensity level of 30% was used as a threshold. Cav3 indicates caveolin 3; ClC, chloride channel; SWELL1, also known as LRRC8A (leucine rich repeat containing protein 8A).

Article Snippet: Masson's trichrome staining and double‐immunolabeling for ClC‐2 (goat polyclonal; SAB2501373, Sigma‐Aldrich), ClC‐3 (rabbit polyclonal; ACL‐001, Alomone Labs), SWELL1 (anti‐LRRC8A, rabbit polyclonal; AAC‐001, Alomone Labs), and Cav3 (mouse monoclonal; 610421, BD Biosciences; rabbit polyclonal; ab2912, Abcam) were performed on paraffin‐embedded sections of unstretched PV preparations.

Techniques: Expressing, Scaffolding

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Caveolae‐Mediated Activation of Mechanosensitive Chloride Channels in Pulmonary Veins Triggers Atrial Arrhythmogenesis

doi: 10.1161/JAHA.119.012748

Figure Lengend Snippet: Co‐IP Western blots showing degree of association of Cav3 and chloride channels ClC‐2, ClC‐3, and SWELL 1 in rat pulmonary vein ( PV ; n=2). IB indicates immunoblot; IP , immunoprecipitation. Cav3 indicates caveolin 3; ClC, chloride channel; SWELL1, also known as LRRC8A (leucine rich repeat containing protein 8A).

Article Snippet: Masson's trichrome staining and double‐immunolabeling for ClC‐2 (goat polyclonal; SAB2501373, Sigma‐Aldrich), ClC‐3 (rabbit polyclonal; ACL‐001, Alomone Labs), SWELL1 (anti‐LRRC8A, rabbit polyclonal; AAC‐001, Alomone Labs), and Cav3 (mouse monoclonal; 610421, BD Biosciences; rabbit polyclonal; ab2912, Abcam) were performed on paraffin‐embedded sections of unstretched PV preparations.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Caveolae‐Mediated Activation of Mechanosensitive Chloride Channels in Pulmonary Veins Triggers Atrial Arrhythmogenesis

doi: 10.1161/JAHA.119.012748

Figure Lengend Snippet: Expression of chloride channels ClC‐2, ClC‐3, and SWELL 1, caveolar scaffolding protein caveolin 3 (Cav3), and potassium channel K ir 2.1 along the pulmonary vein ( PV ) vs left atrium ( LA ). A , Protein expression levels of K ir 2.1, ClC‐2, Cav3, ClC‐3, and SWELL 1 measured in PV distal ( PV dis ), PV ostium ( PV ost ), and LA from the same rat (n=4). B through F , Corresponding protein expression levels normalized to GAPDH (n=4 per group). ** P <0.01 by 1‐way ANOVA with Bonferroni correction. G , Comparative analysis of mRNA expression levels for sarcolemmal chloride ion channel isoforms normalized to GAPDH . n=6 per region for SWELL 1 and n=5 per region for ClC‐2, ClC‐3, and ANO 1. *** P <0.01 vs PV dis for SWELL 1; ## P <0.01, ### P <0.001 vs SWELL 1 for PV dis ; $$ P <0.01 vs ClC‐3 for PV dis ; and && P <0.01 vs ClC‐3 for LA by 2‐way ANOVA with Bonferroni correction. ANO 1 indicates anoctamin 1; Cav3, caveolin 3; ClC, chloride channel; SWELL1, also known as LRRC8A (leucine rich repeat containing protein 8A).

Article Snippet: Masson's trichrome staining and double‐immunolabeling for ClC‐2 (goat polyclonal; SAB2501373, Sigma‐Aldrich), ClC‐3 (rabbit polyclonal; ACL‐001, Alomone Labs), SWELL1 (anti‐LRRC8A, rabbit polyclonal; AAC‐001, Alomone Labs), and Cav3 (mouse monoclonal; 610421, BD Biosciences; rabbit polyclonal; ab2912, Abcam) were performed on paraffin‐embedded sections of unstretched PV preparations.

Techniques: Expressing, Scaffolding

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Caveolae‐Mediated Activation of Mechanosensitive Chloride Channels in Pulmonary Veins Triggers Atrial Arrhythmogenesis

doi: 10.1161/JAHA.119.012748

Figure Lengend Snippet: Molecular and structural remodeling of caveolar mechanosensitive complex. A , Immunoblots for ClC‐2, ClC‐3, SWELL 1, and Cav3 from Wistar ( WT ; n=4) and spontaneously hypertensive rat ( SHR ; n=4) pulmonary vein distal ( PV dis ) tissues. Expression levels were normalized to GAPDH . P values were determined by unpaired Student t test. B , Downregulation of Cav3 correlates with elevated membrane tension and disruption of caveolae structures during hypertension. Representative composite electron micrographs showing the lateral sarcolemmal membranes of cardiomyocytes from nonstretched WT and SHR PV dis tissues. Blue arrowheads denote caveolae connected to plasma membrane (scale bars=200 nm). C and D , Quantification of caveolae density, normalized to membrane length ( C ), and membrane convolution index (L/L o −1) ( D ), n=40 cells per group. Box plots show medians with interquartile range; whiskers represent fifth and 95th percentile; each point represents 1 micrograph. P values were determined by unpaired Student t test. Cav3 indicates caveolin 3; ClC, chloride channel; L, length of membrane contour; L o , shortest length connecting end points of membrane segment; SWELL1, also known as LRRC8A (leucine rich repeat containing protein 8A).

Article Snippet: Masson's trichrome staining and double‐immunolabeling for ClC‐2 (goat polyclonal; SAB2501373, Sigma‐Aldrich), ClC‐3 (rabbit polyclonal; ACL‐001, Alomone Labs), SWELL1 (anti‐LRRC8A, rabbit polyclonal; AAC‐001, Alomone Labs), and Cav3 (mouse monoclonal; 610421, BD Biosciences; rabbit polyclonal; ab2912, Abcam) were performed on paraffin‐embedded sections of unstretched PV preparations.

Techniques: Western Blot, Expressing

Journal: Skeletal Muscle

Article Title: A role for RNA post-transcriptional regulation in satellite cell activation

doi: 10.1186/2044-5040-2-21

Figure Lengend Snippet: Sdc4 −/− satellite cell gene expression post-muscle injury is similar to freshly isolated satellite cells. Myofiber-associated satellite cells are immunoreactive for MyoD 24 h and 48 h after isolation from wild type mice but not Sdc4 −/− mice ( A , B ). Wild type but not Sdc4 −/− cells divide by 48 h in culture ( B ) where c-met (red), MyoD (green), and DAPI (blue) identify satellite cells and a dashed line indicates the position of the myofiber membrane ( A , B ). Flow cytometry histograms of wild type ( C ) and syndecan-4 null ( D ) mononuclear cells from uninjured and injured skeletal muscle 12 h and 48 h post-injury plotted for cell size (FSC) vs. internal complexity (SSC), where the red box indicates gating for further analysis to remove debris (upper panels). Syndecan-3 immunoreactive cells present in the gate were isolated from wild type mice (C, lower panel) and Sdc4 −/− mice (D, lower panel) where the percentages indicate satellite cells (blue lines) relative to other events with false-positives set to an antibody background < 0.1% (red lines). A hierarchical dendrogram constructed with Spotfire™ DecisionSite using Affymetrix GeneChip data reveals that Sdc4 −/− satellite cells cluster most closely to freshly isolated wild type satellite cells while injured wild type satellite cells either 12 h post-injury or 48 h post-injury cluster independently ( E ). Red depicts high relative gene expression and green depicts low relative expression in the hierarchical cluster dendrograms (UPGMA, Euclidean distance). FI, freshly isolated; PI, post-injury.

Article Snippet: Primary antibodies were rabbit anti-cmet (Santa Cruz) at 1:100, mouse anti-MyoD (Novocastra) at 1:10, mouse anti-Pax7 at 1:5 (Developmental Studies Hybridoma Bank), and rabbit anti-MyoD C-20 at 1:500 (Santa Cruz Biotechnology).

Techniques: Expressing, Isolation, Flow Cytometry, Construct

Journal: Skeletal Muscle

Article Title: A role for RNA post-transcriptional regulation in satellite cell activation

doi: 10.1186/2044-5040-2-21

Figure Lengend Snippet: Inhibition of candidate miRNAs alters satellite cell fate. The four candidate miRNAs (miR-16, miR-93, miR-106b, and miR-124) that displayed dynamic expression in satellite cells were inhibited in myofiber-associated satellite cells prior to the first cell division. Transfected cells were assessed 3 or 5 days post myofiber harvest and identified via immunofluorescence as satellite cells by Pax7 expression ( A , B ) with proliferating satellite cells expressing both Pax7 and MyoD ( C , D ) and quiescent satellite cells expressing only Pax7 ( E , F ). The percent of Pax7+ satellite cells decreased between 3 and 5 days in satellite cell populations treated with a scrambled RNA control, however, the relative number of Pax7+ satellite cells remained at similar levels when any candidate miRNA was inhibited ( A , B ). This increase in satellite cells following miRNA inhibition at 5 days was observed in both proliferating satellite cells ( D ) and quiescent satellite cells ( F ) for miR-16, miR-106b, and miR-124 while inhibition of miR-93 resulted in a specific increase in proliferating satellite cells at 5 days ( D ). Inhibition of two miRNAs, miR-106b and miR-124, resulted in a dramatic increase in quiescent satellite cells by 3 days post myofiber isolation ( E ) that remains consistent through 5 days post isolation ( F ).

Article Snippet: Primary antibodies were rabbit anti-cmet (Santa Cruz) at 1:100, mouse anti-MyoD (Novocastra) at 1:10, mouse anti-Pax7 at 1:5 (Developmental Studies Hybridoma Bank), and rabbit anti-MyoD C-20 at 1:500 (Santa Cruz Biotechnology).

Techniques: Inhibition, Expressing, Transfection, Immunofluorescence, Isolation

Journal: International Journal of Molecular Medicine

Article Title: Transcription factor EB-mediated autophagy promotes dermal fibroblast differentiation and collagen production by regulating endoplasmic reticulum stress and autophagy-dependent secretion

doi: 10.3892/ijmm.2020.4814

Figure Lengend Snippet: TGF-β1-induced COL I secretion from fibroblasts is associated with an autophagy-based pathway. Fibroblasts were treated with 50 nmol/l TFEB-siRNA or NC-siRNA for 48 h and then with 10 ng/ml TGF-β1 for 48 h. (A) Fluorescence for colocalization of Rab8a with LAMP1 and COL I (scale bars, 10 or 30 µ m). (B) Line tracing analysis of fluorescence signal intensity. (C) Pearson's colocalization coefficient for COL I and LAMP1. (D) Pearson's colocalization coefficient for COL I and Rab8a. (E) Pro-COL Iα1 secretion in the supernatant of fibroblasts was assessed by enzyme-linked immunosorbent assay. Results are presented as the mean ± SD (n=6). ** P<0.01 vs. control group; ## P<0.01 vs. TGF-β1 treatment group. TGF-β1, transforming growth factor-β1; TFEB, transcription factor EB; siRNA, small interfering RNA; NC, negative control; COL I, collagen I; Rab8a, Ras-related protein Rab-8A; LAMP1, lyso-some-associated membrane protein 1; ns, not significant compared with TGF-β1 treatment group.

Article Snippet: Subsequently, cells were incubated with the following primary antibodies overnight at 4°C: Anti-TFEB (1:50; cat. no. ab220695; Abcam), anti-LAMP1 (1:200; cat. no. ab62562; Abcam), anti-COL I (1:200; cat. no. ab6308; Abcam) and anti-Ras-related protein Rab-8A (Rab8a; 1:200; cat. no. ABIN6290618; Beijing 4A Biotech Co., Ltd.).

Techniques: Fluorescence, Enzyme-linked Immunosorbent Assay, Small Interfering RNA, Negative Control