Article Title: The fungicide ciclopirox inhibits lymphatic endothelial cell tube formation by suppressing VEGFR-3-mediated ERK signaling pathway
Figure Lengend Snippet: CPX does not alter mRNA expression, but inhibits protein synthesis and promotes protein degradation of VEGFR-3 A, CPX did not affect VEGFR-3 mRNA level. Total RNA was extracted from LECs treated with CPX (0-5 μM) for 24 h (Left panel) or with CPX (5 μM) for 0-24 h (Right panel), followed by semi-quantitative RT-PCR. β-actin was used as a loading control. B, CPX inhibited protein synthesis of VEGFR-3 in LECs. LECs were pretreated with CPX (0-5 μM) for 24 h (Left panel) or with CPX (5 μM) for 0-24 h (Right panel), and then pulsed with 35 S-Met/Cys for 4 h, followed by immunoprecipitation with antibodies to VEGFR-3. The immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membranes, followed by autoradiography. GAPDH served as an internal control. C, CPX promoted protein degradation of VEGFR-3 in LECs. LECs, grown in 10% FBS-DMEM medium, were exposed to cycloheximide (CHX, 50 μg/ml), in the presence or absence of CPX (5 μM), for 0-12 h, followed by Western blot analysis with indicated antibodies.
Article Snippet: Human embryonic kidney (HEK) 293 (American Type Culture Collection, Manassas, VA), 293TD and 293A cells (Invitrogen, Carlsbad, CA, USA) were grown in antibiotic-free Dulbecco’s modified Eagle medium (DMEM) (Mediatech) supplemented with 10% heat-inactivated FBS and non-essential amino acid (Mediatech) at 37°C and 5% CO2 .
Techniques: Expressing, Quantitative RT-PCR, Immunoprecipitation, SDS Page, Autoradiography, Western Blot