Cotreatment with Src inhibitors and UCN-01 inhibits both production of multiple angiogenic factors by MM cells and in vitro tube formation of HUVECs. (A) MM.1S cells were washed twice with serum-free medium and then cultured for 24 hours in fresh RPMI 1640 medium containing 10% FBS, after which the supernatants (MM.1S medium) were prepared by centrifuging twice at 500g for 10 minutes each. Control (fresh RPMI 1640 medium with 10% FBS) and MM.1S media were then subjected to the angiogenesis antibody array. According to the table of corresponding factors (supplemental Figure 5C), the angiogenic factors expressed in MM.1S medium, compared with control medium, are highlighted by black squares. (B) MM.1S cells were incubated (16 hours) with 1μM BMS354825 or SKI-606 ± 100nM UCN-01, after which the same array was performed to assess the effects of the drug treatments on the production of angiogenic factors. (C) In parallel, whole cells were lysed and subjected to Western blot analysis to monitor the expression of corresponding factors. The blots were quantified by determining integrated density. Values indicate fold changes for drug-treated cells compared with untreated cells. WCL, Whole-cell lysate; ns, nonspecific bands. (D) Primary HUVECs were preincubated with the cell-permeable dye Calcein AM, and then seeded and incubated (2 hours) with 0.1μM BMS354825 ± 100nM UCN-01 using reduced growth factor basement membrane matrix. Images were captured using an inverted fluorescent microscope (10× magnification). Results are representative of triplicate experiments.