short hairpin rna  (Santa Cruz Biotechnology)

Journal: Molecular cancer therapeutics

Article Title: FAK inhibition disrupts a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth

doi: 10.1158/1535-7163.MCT-13-1063

Figure Lengend Snippet: Association between OPN, β5 integrin, and FAK activation in serous ovarian cancer. A, Kaplan-Meier analyses of integrin β5, αv, β1, β3, FAK, and OPN mRNA levels in 1038 patient samples. High (red) versus low (black) mRNA expression shows patient overall survival probability over 120 months. Hazard ratio (HR) and logrank P significance values are shown (inset). B, representative immunohistochemical staining of sections obtained from paraffin-embedded normal ovary, serous Stage I, and serous Stage II ovarian tumor tissue arrays using antibodies to pY397 FAK, total FAK, β5 integrin, and OPN. Scale is 100 μm. C, β5 integrin staining intensity (0–4) in annotated ovarian tissue arrays. Values are means (+/− SEM, * p < 0.05, n= sample number).

Article Snippet: HEY cells were transduced with lentiviral shRNAs targeting human β5 integrin or Scr shRNA (Mission, Sigma).

Techniques: Activation Assay, Expressing, Immunohistochemical staining, Staining

Journal: Molecular cancer therapeutics

Article Title: FAK inhibition disrupts a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth

doi: 10.1158/1535-7163.MCT-13-1063

Figure Lengend Snippet: Identification of FAK inhibitor sensitive and resistant ovarian carcinoma cells. A, the indicated ovarian carcinoma cell lines were evaluated for anchorage-independent growth over 72 h in DMSO (control) or increasing concentrations of VS-4718 (0.1 to 1.0 μM). Values are means (+/− SEM) of triplicate points from three independent experiments (*** p<0.001 versus control). B, lysates of the indicated cells cultured in suspension with DMSO or VS-4718 (1 μM) for 72 h were analyzed by immunoblotting for pY397 FAK, pS473 AKT, and β5 integrin levels. Corresponding levels of total FAK, Akt, and GAPDH are shown. BT474 breast carcinoma cells are a positive control for pS473 AKT.

Article Snippet: HEY cells were transduced with lentiviral shRNAs targeting human β5 integrin or Scr shRNA (Mission, Sigma).

Techniques: Cell Culture, Western Blot, Positive Control

Journal: Molecular cancer therapeutics

Article Title: FAK inhibition disrupts a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth

doi: 10.1158/1535-7163.MCT-13-1063

Figure Lengend Snippet: FAK inhibition reduces β5 integrin and OPN levels in ID8-IP and HEY cells. A, lysates of ID8 and ID8-IP cells grown in suspension for 72 h immunoblotted for pY397 FAK, total FAK, β5 integrin, OPN, and actin. B, lysates of DMSO- or VS-4718-treated ID8-IP cells grown in suspension for 72 h immunoblotted for pY397 FAK, total FAK, β5 integrin, OPN, and actin. C, DMSO- or VS-4718-treated HEY cells in suspension for 72 h were immunoblotted for pY397 FAK, FAK, Src pY416, c-Src, β5 integrin, OPN, and actin. D, conditioned media from anchorage-independent 24 h DMSO- or VS-4718-treated HEY cells were immunoblotted for OPN and fibronectin. E, stable lentiviral scrambled (Scr, gray) or FAK shRNA (white) knockdown HEY cells were transduced to express GFP, GFP-FAK-WT (green), or GFP-FAK KD (red) and analyzed by flow cytometry. Black histogram, parental HEY background fluorescence. F, HEY cells knocked down and reconstituted with FAK were immunoblotted for exogenous GFP-FAK (~150 kDa) and endogenous FAK (~115 kDa) pY397 FAK and total FAK. Actin is a loading control. G–I, growth of Scr shRNA (gray), FAK shRNA, (white), GFP-FAK WT- (green), and GFP-FAK KD-reconstituted (red) HEY cells in adherent (G), suspended (H), and soft agar (I) growth conditions at 72 hr. Values are means (+/− SD) of triplicate points (***p<0.001) from at least two independent experiments.

Article Snippet: HEY cells were transduced with lentiviral shRNAs targeting human β5 integrin or Scr shRNA (Mission, Sigma).

Techniques: Inhibition, shRNA, Flow Cytometry, Fluorescence

Journal: Molecular cancer therapeutics

Article Title: FAK inhibition disrupts a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth

doi: 10.1158/1535-7163.MCT-13-1063

Figure Lengend Snippet: Genetic FAK inhibition prevents HEY tumor growth associated with decreased OPN and β5 integrin levels. A, mean subcutaneous tumor volume of Scr shRNA (gray, n=6), FAK shRNA (white, n=6), GFP-FAK WT- (green, n=5), and GFP-FAK KD-reconstituted (red, n=6) HEY cells at day 16 to 24 (+/− SD, * p< 0.05, ** p< 0.01; *** p<0.0001). B, final mean subcutaneous tumor mass in panel A (+/− SD, ** p< 0.01). C, Mean GFP-FAK WT (green, n=8), and GFP-FAK KD (red, n=8) HEY orthotopic tumor mass (+/− SD, *p< 0.05). D, representative orthotopic tumors (T) and peritoneal metastasis sites (M) as determined by GFP fluorescent imaging. Scale is 0.5 cm. E, lysates from four GFP-FAK WT or four GFP-FAK KD HEY orthotopic tumors analyzed by pY397 FAK, total FAK, OPN, and actin immunoblotting. F, fluorescent microscopic images of GFP-FAK WT and GFP-FAK-KD HEY tumor sections stained for αvβ5 integrin (red) and cell nuclei (blue). Scale is 100 μm.

Article Snippet: HEY cells were transduced with lentiviral shRNAs targeting human β5 integrin or Scr shRNA (Mission, Sigma).

Techniques: Inhibition, shRNA, Imaging, Western Blot, Staining

Journal: Molecular cancer therapeutics

Article Title: FAK inhibition disrupts a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth

doi: 10.1158/1535-7163.MCT-13-1063

Figure Lengend Snippet: HEY β5 integrin knockdown impairs soft agar and tumor growth with reduced FAK Y397 phosphorylation in spheroids. A, stable HEY β5 integrin knockdown by lentiviral shRNAs (β5-1 and β5-2) as determined by immunoblotting with actin as a loading control. B, flow cytometry of cell surface αvβ5 levels in Scr, β5-1 and β5-2 HEY cells. Black histogram is secondary antibody only. C, mean adherent HEY growth over 4 days (+/− SD, * p<0.05). D, Mean Scr, β5-1 and β5-2 HEY soft agar colony growth over 7 days (+/− SD, * p<0.05, *** p<0.001). E, representative spheroid fluorescent staining for pY397 FAK (red), OPN (green), and cell nuclei (blue) of Scr and β5-1 shRNA HEY cells. Scale is 50 μm. F, orthotopic tumor growth of Scr (n=7), β5-1 (n=9), and β5-2 (n=10) HEY cells in the ovarian bursa. Values are mean tumor mass after 21 days (+/− SD, * p<0.05, ** p<0.01). G, serum from Scr and β5-1 HEY tumor-bearing mice was analyzed by anti-hOPN immunoblotting. Densitometry was used to determine mean values (n=7 tumors each, +/− SD, * p<0.05).

Article Snippet: HEY cells were transduced with lentiviral shRNAs targeting human β5 integrin or Scr shRNA (Mission, Sigma).

Techniques: Western Blot, Flow Cytometry, Staining, shRNA

Journal: Molecular cancer therapeutics

Article Title: FAK inhibition disrupts a β5 integrin signaling axis controlling anchorage-independent ovarian carcinoma growth

doi: 10.1158/1535-7163.MCT-13-1063

Figure Lengend Snippet: Stable activated-Akt (Akt*) expression in HEY and OVCAR8 cells promotes anchorage-independent growth and β5 integrin surface expression in the presence of VS-4718. A, immunoblotting for pY397 FAK, FAK, pS473 Akt, pT308 Akt, Akt, and GAPDH in control vector (CTRL) or Akt*-expressing HEY or OVCAR8 cells cultured in suspension for 72 h with 1 μM VS-4718 as indicated. B, HEY and OVCAR8 non-transfected (NT), CTRL-, and Akt*-expressing cells were evaluated for anchorage-independent growth over 72 h in DMSO or 1 μM VS-4718. C, OVCAR10 cells plated in suspension for 72h and treated with DMSO (D), 0.1 μM VS-4718 (VS), or 0.1 μM Wortmannin (W) alone or in combination. Right, immunoblotting for pS473 Akt, Akt, and actin. B and C, values are means (+/− SEM) of triplicate points from three independent experiments (* p<0.05, ** p<0.01, *** p<0.001). D, flow cytometry of αvβ5 surface expression in CTRL or Akt* HEY cells cultured in DMSO or 1 μM VS-4718 for 72 h. Dark histogram is secondary antibody only.

Article Snippet: HEY cells were transduced with lentiviral shRNAs targeting human β5 integrin or Scr shRNA (Mission, Sigma).

Techniques: Expressing, Western Blot, Plasmid Preparation, Cell Culture, Transfection, Flow Cytometry