plko 1 control shrna vector targeting gfp  (Addgene inc)

Journal: Molecular Cancer

Article Title: IL-6 expression predicts treatment response and outcome in squamous cell carcinoma of the esophagus

doi: 10.1186/1476-4598-12-26

Figure Lengend Snippet: Role of IL-6 in tumor growth. a . Effect of an IL-6-GFP silencing vector on the level of IL-6 in CE81T, as assessed by immunofluorescence and immunoblotting. The results of representative slides are shown. The IL-6-GFP silencing vector as compared with that with control-GFP vector significantly reduced IL-6 levels. b . Effect of IL-6 on the proliferation of CE81T cancer cells as determined by viable cell counting. The same number of cells (10 4 ) were plated in each plate on day 0 and allowed to grow in their respective cultures. We counted the number of viable cells after incubation for 2, 4 and 6 days. The Y axis represents the viable cell number. Point, means of three separate experiments; bars, SD. *, p < 0.05. c . Effect of IL-6 inhibition on tumor xenograft growth. Data represent the means ± SD of three independent experiments. *, p < 0.05. IL-6 expression was also evaluated by IHC staining of xenografts. Representative slides are shown. d . Effect of IL-6 inhibition on cell death, as assessed by FACS and immunofluorescence staining. The results of representative slides are shown.

Article Snippet: The IL-6-GFP silencing vector (human IL6 shRNA constructs in retroviral GFP vector) and GFP-control vector (Non-effective scrambled shRNA cassette in retroviral GFP vector) were purchased from Origene Technologies, Inc. (Rockville, MD).

Techniques: Plasmid Preparation, Immunofluorescence, Western Blot, Cell Counting, Incubation, Inhibition, Expressing, Immunohistochemistry, Staining

Journal: Molecular Cancer

Article Title: IL-6 expression predicts treatment response and outcome in squamous cell carcinoma of the esophagus

doi: 10.1186/1476-4598-12-26

Figure Lengend Snippet: Effects of circulating IL-6 on tumor aggressiveness. a . The levels of IL-6 in cell supernatant and serum of mice bearing tumors with or without IL-6 silencing vectors were examined by ELISA. Columns, means of 3 separate experiments; bars, SD. *, P < 0.05. b . Serum IL-6 levels of patients were examined by ELISA analysis. Columns are the means of specimen; Bars, SD; *, P < 0.05. (LR = Local-regional Recurrence /persistent; DM = distant metastasis). c . IHC using MMP-9, E-cadherin, CD31, and VEGF staining in tumors 20 days after implantation. IL-6 silencing vectors were significantly reduced by the angiogenesis and EMT-related changes as compared with control-GFP vector. The Y-axis represents the ratio normalized by the value of target protein in tumor transfected with control vectors. Columns, means of 3 separate experiments; bars, SD. *, P < 0.05 (C-V, cells transfected with the control vector; IL-6 SV, cells transfected with the IL-6 silencing vector).

Article Snippet: The IL-6-GFP silencing vector (human IL6 shRNA constructs in retroviral GFP vector) and GFP-control vector (Non-effective scrambled shRNA cassette in retroviral GFP vector) were purchased from Origene Technologies, Inc. (Rockville, MD).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Plasmid Preparation, Transfection

Journal: PLoS ONE

Article Title: Chymase Mediates Injury and Mitochondrial Damage in Cardiomyocytes during Acute Ischemia/Reperfusion in the Dog

doi: 10.1371/journal.pone.0094732

Figure Lengend Snippet: (a) Immunofluorescence pattern of HL-1 cells transfected with p-GFP-shNR4A1 and scrambled p-GFP-shRNA for 5 days, loaded with dye (MitoCapture) and then treated with 5 µg/ml chymase (chy) for 2 h. Chymase treatment induces Mt apoptosis that is detected by loss of an electrochemical gradient and the inability to take up the MitoCapture dye. Normal Mt; bright red; Apoptotic Mt: little or no red staining. Bright green is the green fluorescent protein (GFP) and indicates that the cells were transfected with either p-GFP-shNR4A1 or scrambled p-GFP-shRNA. Cells without transfection and chymase treatment are used as the control. (b) Percentage of Mt apoptosis in chymase treated HL-1 cells, HL-1 cells transfected with p-GFP-shNR4A1 and scrambled p-GFP-shRNA. (c) NR4A1 protein expression in normal HL-1 cells, HL-1 cells transfected with p-GFP-shNR4A1 and scrambled p-GFP-shRNA. NL: HL-1 cells without transfection.

Article Snippet: HL-1 cells were transfected with p-GFP-ShNR4A1 and scrambled p-GFP-ShRNA (OriGene, MD) for 5 days and then treated with 5 µg/ml chymase for 2 h. Mitochondrial apoptosis was detected by Abcam Apoptosis Detection Kit (Abcam, MA).

Techniques: Immunofluorescence, Transfection, shRNA, Staining, Expressing