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. 2014 Apr 23;5(8):2030–2043. doi: 10.18632/oncotarget.1918

Figure 3. MDM2 Inhibitor Combinations with PI3K and MEK Inhibitors Yield Dramatic Increases in Apoptosis but Small Incremental Reductions in Proliferation.

Figure 3

(A) CAL-51 or (B) NCI-H460 cells were treated with DMSO (control), 3 μM C-15 (MDM2 inhibitor), (A) 0.3 μM or (B) 3 μM AMG 511 (PI3K inhibitor), or a combination of C-15 plus AMG 511 in the presence of caspase 3/7 substrate for (A) 8 hours or (B) 48 hours. (C) RT4, (D) RKO, (E) A427 or (F) C32 cells were treated with DMSO (control), 3 μM C-15 or (C) 0.3 μM or (D-F) 3 μM trametinib (MEK inhibitor) for (D) 30 hours or (C, E, F) 48 hours as described above. Apoptotic indices were calculated as the percentage of caspase-positive objects relative to the total number of DNA-containing objects; mean and SEM (n=3) are shown. RKO cells were treated with a dose titration matrix (3-fold dilution series) of C-15 (MDM2i) and trametinib (MEKi) for 48 hours and pulsed with bromodeoxyuridine prior to the end of treatment. Cells were trypsinized, fixed, permeabilized, acid-treated, and stained with propidium iodide, anti-BrdU and anti-caspase-3 antibodies. (G) The percentage of sub-G1 cells was measured by flow cytometry for each condition in the dose titration matrix. (H) Representative DNA ploidy plots for cells treated with vehicle, 5 μM C-15, 0.5 μM trametinib, or a combination of C-15 plus trametinib with populations of G1 (red), S (blue), G2/M (light green), sub-G1 (dark green), and caspase-3 positive (magenta) cells shown.