Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Tang-Luo-Ning, a Traditional Chinese Medicine, Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis of Schwann Cells under High Glucose Environment

doi: 10.1155/2017/5193548

Figure Lengend Snippet: TLN can decrease the expression of GADD34 and Ero1α and increase the expression of P-eIF2α in high glucose-induced RSC96 cells. (a) Images of GADD34 relative protein level measured by high content analysis; images were viewed at a magnification of 10x. (b) Images of Ero1 α and P-eIF2 α relative protein level measured by Western blot, normalized to β -actin. ((c)–(e)) Summarized data of GADD34, Ero1 α , and P-eIF2 α , normalized as fold change of 25 mM glucose group. Data were analyzed by one-way ANOVA followed by least significant difference. Data were shown as mean ± SEM ( n = 4). ★★ P < 0.01 and ★ P < 0.05 versus 25 mM glucose; ☆☆ P < 0.01 and ☆ P < 0.05 versus 150 mM glucose.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-Ero1 α (1 : 500, Santa Cruz, sc-100805); rabbit polyclonal anti-IRE1 α (1 : 2000, Santa Cruz, sc-20790), anti-P-IRE1 α (1 : 2000, Abcam, ab48187), and anti-P-eIF2 α (1 : 1500, Santa Cruz, sc-293100).

Techniques: Expressing, High Content Screening, Western Blot

Journal: PLoS ONE

Article Title: Vapor of Volatile Oils from Litsea cubeba Seed Induces Apoptosis and Causes Cell Cycle Arrest in Lung Cancer Cells

doi: 10.1371/journal.pone.0047014

Figure Lengend Snippet: ( A ) Immunoblot analysis of pMdm2 Ser 166 , p21 and p53 was analyzed in control or VCO exposed A549 cells at indicated time periods. β-actin used as internal loading control. ( B ) Immunoblot showed p21 level in p53 siRNA or Sp1 siRNA or their control siRNA transfected A549 cells exposed with or without VCO. ( C ) ChIP assay demonstrated VCO exposure increases binding of p53 to its response element (RE1 and RE2) on p21 promoter. ( D ) Cyclin D1-p21 interaction was increased with increasing the time of VCO exposure, which was shown by co-immunoprecipitation study. ( E ) BrdU incorporation in control and VCO treated A549 cells were examined by florescence microscopy. ( F ) FACS analysis showed cell cycle arrest at G1 to S phase as indicated by increased percentage of G 0 /G 1 cells with the decrease of S and G 2 /M phase cells. Bar represents 20 µm.

Article Snippet: The primary antibodies for pAkt (Thr308; sc-135650), pAkt1/2/3 (Ser473; sc-7985-R), Akt 1/2/3 (sc-8312), pPDK1 (Ser241; sc-101775), Bcl-xL (sc-7195), pBad (Ser136; sc-7999), Bad (sc-7869), pMdm2 (Ser166; sc-293105), p53 (sc-6243), p21 (sc-756), cyclin D1 (sc-753), poly[ADP-ribosyl]-polymerase (PARP) (sc-7150), Cytochrome c (sc- 7159) and β-actin (sc-130657) were purchased from Santa Cruz Biotechnology Inc., California, USA and mTOR (#2983) was procured from Cell Signaling Technology Inc., Danvers, MA, USA.

Techniques: Western Blot, Transfection, Binding Assay, Immunoprecipitation, BrdU Incorporation Assay, Microscopy

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, Filtration

Journal: Molecular cancer research : MCR

Article Title: G9a promotes invasion and metastasis of non-small cell lung cancer through enhancing focal adhesion kinase activation via NF-κB signaling pathway

doi: 10.1158/1541-7786.MCR-20-0557

Figure Lengend Snippet: A. Western blot analysis showing that activation of FAK signal pathway was attenuated by knockdown of FAK by siRNA in G9a-overexpressed NSCLC cells. B. Representative images of scratch assays. 0h and 24h/ 36h for the controls (pcDNA 3.1) and G9a-overexpressed (G9a-pcDNA 3.1) H1299 / A549 upon knockdown of FAK by its specific siRNA, respectively. C. Quantitative analysis of migration distances in the control and G9a-overexpressed A549 and H1299 cells after FAK is attenuated by its specific siRNA. D. Representative images for Matrigel transwell invasion assays of the controls (pcDNA 3.1) and G9a-overexpressed (G9a-pcDNA 3.1) H1299 and A549 transfected with control and FAK siRNA. E. Quantitative analysis for invasive cells in the controls and G9a-overexpressed A549 and H1299 cancer cells upon knockdown of FAK. Data are presented as percentages of the control cells transfected with the empty plasmid pcDNA 3.1 (* P < 0.05, ** P < 0.01, *** P < 0.001, NS represents for no significance; compared to the controls cells).

Article Snippet: A FAK siRNA (Cat#: sc-29310) was purchased from Santa Cruz Company.

Techniques: Western Blot, Activation Assay, Migration, Transfection, Plasmid Preparation