Journal: The American Journal of Pathology

Article Title: Smooth Muscle α-Actin Deficiency Leads to Decreased Liver Fibrosis via Impaired Cytoskeletal Signaling in Hepatic Stellate Cells

doi: 10.1016/j.ajpath.2019.07.019

Figure Lengend Snippet: Cellular features and COL1 expression of smooth muscle (SM) α-actin–deficient hepatic stellate cells (HSCs). A: HSCs from Acta2+/+ and Acta2−/− mice were cultured in standard medium as indicated, and images were captured using darkfield phase microscopy. The arrows indicate an individual HSC, and the insets show a higher magnification (representative images are shown from >10 others). B: Cells were cultured as in A for 3 days and subjected to Oil Red O staining. The arrows indicate cytoplasmic lipid droplets in HSCs (representative images are shown of >10 others). C: Cells were as in A, and total RNA was isolated. Real-time quantitative PCR was performed to detect the indicated mRNAs. D: Cells were cultured as in A for 5 days and then incubated in 0.5% serum medium with or without 10 ng/mL TGF-β or 20 μmol/L ET-1 for 24 hours. Total RNA was isolated, and COL1α1 mRNA levels were measured by real-time quantitative PCR. E: Rat hepatic stellate cells were exposed to Ad-control or Ad-SM α-actin [25 or 50 multiplicity of infection (MOI)] for 3 days in 0.5% serum medium and then incubated in 0.5% serum medium for an additional 1 day; whole-cell lysates were subjected to immunoblotting as indicated, and the quantitative data were presented graphically. n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001 for Acta2+/+ versus Acta2−/−. Scale bars: 20 μm (A); 40 μm (B). Original magnification, ×20 (insets).

Article Snippet: Anti-COL1 antibody (600-401-103-0.5) was from Rockland (Gilbertsville, PA).

Techniques: Expressing, Cell Culture, Microscopy, Staining, Isolation, Real-time Polymerase Chain Reaction, Incubation, Infection, Western Blot

Journal: The American Journal of Pathology

Article Title: Smooth Muscle α-Actin Deficiency Leads to Decreased Liver Fibrosis via Impaired Cytoskeletal Signaling in Hepatic Stellate Cells

doi: 10.1016/j.ajpath.2019.07.019

Figure Lengend Snippet: A proposed molecular mechanism underlying the effect of smooth muscle (SM) α-actin on hepatic myofibroblast differentiation and liver fibrogenesis. SM α-actin is encoded by Acta2 and transactivated by serum response factor (SRF) during hepatic stellate cell activation. This leads to actin polymerization and stress fiber formation, which not only serves as the functional actin cytoskeleton, but also plays an important role in transmission of mechanical signals to the nucleus to regulate COL1 expression. Additionally, signaling mediated by important soluble factors such as transforming growth factor-β (TGF-β) and ET-1 is impaired in Acta2-deficient hepatic stellate cells, contributing to reduced expression of COL1 and liver fibrosis in Acta2-deficient mice.

Article Snippet: Anti-COL1 antibody (600-401-103-0.5) was from Rockland (Gilbertsville, PA).

Techniques: Activation Assay, Functional Assay, Transmission Assay, Expressing

Journal: Biomedicines

Article Title: Cell Type-Specific Anti-Adhesion Properties of Peritoneal Cell Treatment with Plasma-Activated Media (PAM)

doi: 10.3390/biomedicines10040927

Figure Lengend Snippet: PAM treatment attenuates protein biosynthesis and the secretion of pro-adhesive factors. Fibroblasts were incubated with 1:2 diluted PAM for 4 h and analyzed after indicated timepoints. ( A ) Representative IF microscopy after staining with anti-5mC antibodies and ( B ) relative genomic methylation level per nucleus (number of foci normalized to the control); the scale bar equals 10 µm. ( C ) DNMT activity level per cell. ( D ) DigiWest protein profiles of pro-adhesive factors collagen I, FGF receptor and FGF-10 and fibronectin relative to controls. ( E ) Cytokine multiplex assay of GM-CSF and IL-1b in fibroblast supernatants relative to control. ( F , G ) Representative IF microscopy of fibronectin ( F ). Scale bars represent 100 and 10 µm, respectively. ( G , H ) Representative Western blot of fibronectin ( G ), and relative of fibronectin expression ( H ) (analyzed from ( G )). ( I , J ) Representative IF microscopy of collagen I ( I ). Scale bars represent 100 and 10 µm, respectively. ( J , K ) Representative Western blot of collagen I ( J ), and relative collagen I expression ( K ) (analyzed from ( J )). ( L ) Relative extracellular hydroxyproline expression. ( M ) Relative MMP-2 expression. ( N ) Relative expression of TGF β. Results are expressed as mean ± SD; * p < 0.05 as determined by paired t -test.

Article Snippet: After washing, membranes were incubated overnight at 4 °C with primary antibodies under constant agitation: fibronectin (1:1000 in 0.1% BSA, abcam), collagen I-alpha antibody (1:1000 in 0.1% BSA, NovusBiologicals, Littleton, CO, USA) and GAPDH (1:1000 in 0.1% BSA, Cell Signaling 14C10).

Techniques: Incubation, Microscopy, Staining, Methylation, Activity Assay, Multiplex Assay, Western Blot, Expressing