Cotreatment with BMS354825 and UCN-01 inhibits VEGF expression in MM cells in association with disruption of Src function. (A) MM cells were exposed (16 hours) to UCN-01 (U266, 150nM; RPMI 8226, MM.1S, and MM.1R, 100nM) ± BMS354825 (0.5μM), after which ELISA assays were performed to monitor VEGF levels, using a standard curve (supplemental Figure 5B), in cell-culture supernatants. Numbers indicate P values versus untreated controls. (B) MM.1S and U266 cells were treated with UCN-01 (MM.1S, 100nM; U266, 150nM) ± 0.5μM BMS354825 for 6 hours (MM.1S) or 16 hours (U266), after which total RNA was isolated and subjected to quantitative PCR analyses to assess human VEGF-A mRNA levels, as described in the supplemental Methods. VEGF expression was expressed as the fold increase relative to values for untreated controls (arbitrarily set at 1). (In panels A and B, results represent the means ± SD for triplicate determinations performed on 3 separate occasions. (C) MM.1S and U266 cells were incubated with UCN-01 (MM.1S, 100nM; U266, 150nM) ± the indicated concentrations of BMS354825 for 16 hours, after which cells were lysed and subjected to Western blot analysis to detect expression of VEGF. d, dimer; m, monomer. (D) U266 cells transfected with wt c-Src were treated with 150nM UCN-01 ± the indicated concentrations of BMS354825 for 16 hours, after which Western blot analysis was performed to monitor phosphorylation of Src (Y418) and its substrates, including FAK (Y576/577), p130Cas (Y410), and paxillin (Y118), as well as phosphorylation of ERK1/2 and PYK2. Vertical lines have been inserted to indicate repositioned gel lanes. In parallel, expression of intracellular VEGF was detected. In panels C and D, the blots for VEGF monomer were quantified by determining integrated density using a densitometer. Values indicate fold changes for drug-treated cells compared with untreated cells.