sirna focal adhesion kinase (fak  (Cientifica Ltd)

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

Article Title: Myocardial Cytoskeletal Adaptations in Advanced Kidney Disease

doi: 10.1161/JAHA.121.022991

Figure Lengend Snippet: A , Expression of FAK mRNA is significantly upregulated in left ventricular tissues from patients undergoing hemodialysis, ex vivo. Analysis of FAK mRNA expression by quantitative polymerase chain reaction revealed that FAK was significantly upregulated in hearts from patients undergoing hemodialysis. However, FAK mRNA expression was suppressed in hearts from patients with HTN. B , Expression of FAK protein is upregulated in left ventricular tissues from patients undergoing hemodialysis, ex vivo. Protein expression of FAK was significantly upregulated in hearts from patients undergoing hemodialysis as assessed by immunoblotting. FAK protein expression was significantly suppressed in HTN hearts. C , FAK silencing mediates cytoskeletal dysfunction in the presence of mineral stressors. (i) Primary human ventricular cardiac fibroblasts were transfected once with either scrambled or FAK siRNA. Cells were then treated daily with or without mineral stressors (ie P+Ca: 2 mmol/L β‐glycerophosphate with 1 unit/mL alkaline phosphatase, and 2.7 mmol/L calcium chloride) for 5 days. After treatment, target cytoskeletal proteins were assessed by immunoblotting. (ii) Treatment with FAK siRNA significantly reduced full‐length FAK expression at 5 days, and exposure to high calcium and high phosphate further reduced FAK expression. High calcium and high phosphate treatment resulted in detection of 80 kDa N‐terminal FAK fragment, which was decreased in combined FAK siRNA and high mineral stressors treatment. There was no statistical difference in FAK expression between the control and scrambled siRNA groups. (iii) There was no significant difference in β‐actin expression across the groups. (iv) β‐Tubulin was significantly decreased in cardiac fibroblasts exposed to mineral stressors compared with controls, but was not significantly different in the FAK silencing groups. (v) Vimentin was decreased significantly when cardiac fibroblasts were exposed to high calcium and high phosphate, and further decreased in the FAK silenced group exposed to high calcium and high phosphate. (vi) Expression of vinculin followed a similar pattern to vimentin. D , Schematic diagram of ultrastructural adaptations of the failing myocardium in CKD. In the healthy individuals, interactions between the heart and kidney is critical for normal physiological function. Patients with advanced CKD on hemodialysis (HD) exhibited significant cytoskeletal maladaptations that were generally more severe than hypertensive control hearts. HD hearts exhibited a unique pattern of severely reduced β‐actin, β‐tubulin and upregulation of vinculin expression. Cytoskeletal dysregulation in HD and HTN hearts is associated with mitochondrial dysfunction (reduced OPA1 and MFN1 genes responsible for mitochondrial fusion and division) and loss of cell survival pathways. FAK is a cytosolic tyrosine kinase that plays a central role in regulating cytoskeletal integrity. FAK expression is upregulated in HD hearts. This may account for increased expression of anchor protein vinculin observed as a protective response. GFR indicates glomerular filtration rate.

Article Snippet: Human ventricular cardiac fibroblasts were serum starved for 24 hours prior to transfection with either focal adhesion kinase (FAK) siRNA (Cat. No. sc‐29310; Santa Cruz Biotechnology, Dallas, TX) or siRNA‐B (Cat. No. sc‐44230; Santa Cruz Biotechnology, Dallas, TX).

Techniques: Expressing, Ex Vivo, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Control, Filtration

Journal: Clinical Cancer Research

Article Title: Alpha-Smooth Muscle Actin (ACTA2) Is Required for Metastatic Potential of Human Lung Adenocarcinoma

doi: 10.1158/1078-0432.ccr-13-1181

Figure Lengend Snippet: Figure 5. Silencing of ACTA2 suppressed c-MET and FAK expression. A, PC14PE6 cells were transfected with control (siCTRL) or ACTA2 siRNA (siACTA2). At 48 hours after transfection, protein and mRNA levels of ACTA2, c-MET, and FAK were determined by Western blot analysis (left) and qRT-PCR (right), respectively. GAPDH ¼ internal control. B, the protein and mRNA levels of the EMT makers, E-cadherin and vimentin, were determined by Western blot analysis (left) and qRT-PCR (right), respectively. C and D, PC14PE6 cells were transfected with c-MET or FAK siRNA. At 48 hours after transfection, in vitro migration (C) and invasion (D) were determined by wound closure assay and Matrigel Transwell assay, respectively. The data shown are the mean plus SD from 3 separate experiments. , P < 0.05.

Article Snippet: For transfection, human NSCLC cells were plated at a density of 5 10 cells per dish and transfected with the indicated siRNAs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. siRNAs directed againstACTA2, Focal adhesion kinase (FAK), and c-MET were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR, In Vitro, Migration, Wound Closure Assay, Transwell Assay