Figure 4. ATR modulation by shRNAs or using a specific inhibitor (VE-821) reduces MM cell growth.

A, H929 MM cells (left panels, blue) and OPM-2 MM cells (right panels, red) were transiently transfected with a shRNA against ATR or with a scrambled shRNA. Data derived from two independent experiments in triplicates are reported. For each cell line, cellular growth is shown on the left and apoptosis on the right. Cellular growth is measured by cell count with trypan blue exclusion at day 0–3, while apoptosis is evaluated by Annexin V-PE/7-AAD on GFP-gated cells at 48 hours. B, Left panel: Immunofluorescence staining for γ-H2A.X foci was performed in H929 and OPM-2 cells silenced with scrambled and ATR shRNAs at 48 hours. Right panel: Number of γ-H2A.X foci with average ± SD is reported. C, Lysates were obtained at 48 hours after transfection. Western blot analysis using antibodies against ATR, γ-H2A.X, and GAPDH (as loading control) was used. D, Left panel: MTT viability assay was used to evaluate growth inhibitory effects upon incubation for 72 hours with VE-821, with doses ranging from 0 to 10μM. Right panel: Apoptosis was measured with Annexin V-FITC/PI staining, upon incubation with DMSO, 1 μM, and 2.5 μM for 48 hours. Data are obtained from two independent experiments, performed in triplicate. E, western blot analysis for total PARP, γ-H2A.X, and GAPDH after treatment with DMSO, 1 μM, and 2.5 μM for 48 hours. F, U266 cells were transfected with MYC-EGFP and LACZ-EGFP and then treated with 5μM VE-821 for 48 h, starting from Day 0 of transfection. Annexin V-PE/7-AAD staining was used to measure the percentage of dead cells. G, H929 and MM.1S myeloma cells were silenced with shRNAs targeting c-MYC and scrambled and then treated with 2.5 μM VE-821 for 48 hours. Apoptosis was evaluated as in (F).