Journal: Cancer Research

Article Title: Down-regulation of Vascular Endothelial Growth Factor Receptor 2 Is a Major Molecular Determinant of Proteasome Inhibitor–Mediated Antiangiogenic Action in Endothelial Cells

doi: 10.1158/0008-5472.can-08-3150

Figure Lengend Snippet: Figure 1. Effects of proteasome inhibitors on the VEGFR2 protein expression in HUVECs. Representative Western blot analyses of endothelial cells that were left untreated (solvent only) or treated with lactacystin (1 Amol/L), MG132 (1 Amol/L), and ALLN (10 Amol/L) in a time-dependent manner (A) as indicated and lactacystin, MG132, and ALLN in a concentration- dependent manner (B) as indicated. Total cellular protein was separated by 8% SDS-PAGE. VEGFR2, VEGFR1, tie-2, and tubulin proteins were detected by enhanced chemiluminescence. Comparable results were obtained from three independent experiments. C, effects of MG132 and ALLN on the soluble VEGFR2 expression in supernatants of HUVEC. Soluble VEGFR2 protein content was assayed in culture supernatants by sVEGFR2 ELISA (R&D Systems) according to the manufacturer’s instructions. HUVECs were left untreated (solvent only, DMSO 0.1%) or treated with MG132 (at 1 mmol/L) and ALLN (at 10 Amol/L). Columns, mean values from triplicate experiments; bars, SE (Student’s t test). n.s., not significant.

Article Snippet: The membranes were incubated with the indicated primary antibodies [VEGFR1 (MAB321, clone49560), VEGFR2 (MAB3573, clone89109), and tie-2 (MAB313, clone83711) from R&D Systems; Sp1 (clone PEP2) and flt-1 (clone H-225) from Santa Cruz; tubulin antibody from LabVision], followed by incubation with horseradish peroxidase–conjugated secondary antibodies (anti-mouse or anti-rabbit IgG, Amersham; anti-goat, Dako).

Techniques: Expressing, Western Blot, Solvent, Concentration Assay, SDS Page, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Identification of Cross Talk between FoxM1 and RASSF1A as a Therapeutic Target of Colon Cancer

doi: 10.3390/cancers11020199

Figure Lengend Snippet: Exogenous VEGF 165 suppresses RASSF1A expression in normal epithelial and endothelial cells. Metastatic colon cancer cells (mCRC, ( A ) T84 and ( B ) Colo 205) were stimulated with VEGF 165 as indicated before and cell metabolism was performed by MTT assay for 6 h (black dash), 24 h (green) and 48 h (pink). Immortalized benign protate (( C ) RWPE1), human bronchial epithelial cells (( D ) HBEpc), immortalized human breast epithelial cells (( E ) MCF-10A), endothelial cells ( F , EC) were stimulated with or without VEGF 165 (12.5 and 25 ng/mL) for 24 h. Cell lysates were monitored by Western blot for p-VEGFR1, p-VEGFR2, RASSF1A, and GAPDH as indicated. Immunoblot was quantified by scanning densitometry and normalized against GAPDH expression for RWPE1 ( G ), HBEpc ( H ), MCF 10A ( I ) and EC (lower panel of ( F )). Results are from three independent experiments and statistical significance was determined using one way-ANOVA followed Bonferroni test. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The primary antibodies used were against RASSF1A (eBioscience, San Diego, CA, USA, #14-6888-80), p-YAP (Cell Signaling, Danvers, MA, USA Ser127, #D9W21), YAP (G-6) (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-376830), p-Akt (thr308) (Cell Signaling, #9275), total Akt (Cell Signaling, #9272), p-VEGFR1/Flt-1 (Y1213) (R and D Systems, #AF4170), p-VEGFR2 /KDR/Flk-1 (Y1214) (R and D Systems, Minneapolis, MN, USA, #AF1766), total VEGFR1 (R&D systems, #AF321), total VEGFR2 (R&D systems, #AF357), PTEN (R and D Systems, AF847), FoxM1 (R and D Systems, #AF3975), GAPDH (Sigma Aldrich, St. Louis, MO, USA, #G8795).

Techniques: Expressing, MTT Assay, Western Blot

Journal: Cancers

Article Title: Identification of Cross Talk between FoxM1 and RASSF1A as a Therapeutic Target of Colon Cancer

doi: 10.3390/cancers11020199

Figure Lengend Snippet: Neutralizing VEGF receptor antibodies and Akt inhibition upregulates RASSF1A and downregulates FoxM1 in mCRC. T84 and Colo 205 cells were treated with VEGF receptor 1 and VEGFR2 as indicated before (see ). Protein expression from cell lysates was determined by immunoblotting for RASSF1A (( A ), upper panel) and FoxM1 (( A ), middle panel). Densitometry analysis was performed and normalyzed with GAPDH expression to demonstrate significant upregulation for RASSF1A ( B ) and downregulation of FoxM1 in the presence of neutralized (NT) VEGFR1 or VEGFR2 ( C ). ( D , E ) T84 and Colo 205 cells were treated with Akt inhibitor (wortmannin) with different doses (0–1 µM) for 24 h. Cell lysates were analyzed for pAkt (1st lane), total Akt (2nd lane), RASSF1A (3rd lane) expression by immunoblot analysis and quantified by densitometry ( F , G ). The results are from three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The primary antibodies used were against RASSF1A (eBioscience, San Diego, CA, USA, #14-6888-80), p-YAP (Cell Signaling, Danvers, MA, USA Ser127, #D9W21), YAP (G-6) (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-376830), p-Akt (thr308) (Cell Signaling, #9275), total Akt (Cell Signaling, #9272), p-VEGFR1/Flt-1 (Y1213) (R and D Systems, #AF4170), p-VEGFR2 /KDR/Flk-1 (Y1214) (R and D Systems, Minneapolis, MN, USA, #AF1766), total VEGFR1 (R&D systems, #AF321), total VEGFR2 (R&D systems, #AF357), PTEN (R and D Systems, AF847), FoxM1 (R and D Systems, #AF3975), GAPDH (Sigma Aldrich, St. Louis, MO, USA, #G8795).

Techniques: Inhibition, Expressing, Western Blot