PP2A activation induced p21-mediated cell death and differentiation via c-Myc degradation. (A) HL-60 and MV4-11 cells treated with Veh or 2.5 μM OSU-2S resulted in increased p21 levels as evidenced by immunoblotting, representative images. Relative densitometric quantification is presented below the blots, as well as in the graph of p21 protein levels (B), normalized to the actin loading control. OSU-2S treatment significantly increased p21 protein expression. (C) Densitometric quantification of p21 immunoblotting, normalized to the actin loading control, and western blot images of primary AML cells treated with OSU-2S (3.5 μM, 16 hours). OSU-2S significantly induced p21 expression in primary AML. (D) Cells treated with OSU-2S (1.75 µM [HL-60] or 2.5 µM [MV4-11] for 8 hours) 24 hours after transfection with negative control (nc) or PP2Ac siRNA were probed for p21 expression by immunoblot analysis; representative images are shown. Relative densitometric quantification is presented as numbers below the blots, as well as in the graph of relative p21 protein levels (E), normalized to the actin loading control. OSU-2S–mediated p21 induction was significantly reversed by PP2Ac siRNA, confirming involvement of PP2A. (F) Relative viability (annexin V/PI) in cells transfected with nc siRNA or p21 siRNA (60 nM) and treated 24 hours after transfection with Veh and 1.75 µM OSU-2S (HL-60) or 2.5 µM OSU-2S (MV4-11, U937) for an additional 24 hours. p21 knockdown significantly rescued OSU-2S–mediated cytotoxicity (HL-60: n = 15, P < .0001, 70.3% rescue; MV4-11: n = 7, P < .0001, 58.5% rescue, U937: n = 5, P = .0007, 50.24% rescue). Immunoblot analysis (48 hours after transfection) confirmed p21 knockdown. (G) Percent CD11b ⁺ cells in HL-60 or U937 cells transfected with nc or p21 siRNA and treated 24 hours after transfection with Veh or OSU-2S (1.75 µM, 16-24 hours). p21 knockdown significantly reversed OSU-2S–mediated differentiation (HL-60: n = 12, P < .0001, 86.96% rescue; U937: n = 5, P = .0053, 94.29% rescue). (H) OSU-2S treatment (2.5 μM) resulted in c-Myc downmodulation in HL-60 and MV4-11 cells. (I) Representative western blot images and densitometric quantification of c-Myc protein levels, normalized to actin. (J) OSU-2S treatment (3.5 μM, 16 hours) resulted in c-Myc downmodulation in primary AML cells; western blot images and densitometric quantification. (K) Cells treated with OSU-2S (1.75 µM [HL-60] or 2.5 µM [MV4-11] for 8 hours) 24 hours after transfection with nc or PP2Ac siRNA were probed for c-Myc expression. (L) Representative western blot images and densitometric quantification of c-Myc, normalized to actin. OSU-2S–mediated c-Myc downregulation was significantly reversed by PP2A siRNA, as seen by immunoblot analysis, confirming involvement of PP2A. (M) Cells treated with OSU-2S (1.75 µM [HL-60] or 2.5 µM [MV4-11] for 8 hours) 24 hours after transfection with nc or B56α siRNA were probed for c-Myc expression. Representative western blot images and (N) densitometric quantification of c-Myc, normalized to actin. OSU-2S–mediated c-Myc downregulation was significantly reversed by PP2A B56α siRNA according to immunoblot analysis. (O) Percentage of relative viability (annexin V/PI) in cells transfected with empty pcDNA3 vector or pcDNA3-c-Myc and treated 48 hours after transfection with Veh and 1.75 µM OSU-2S (HL-60) or 2.5 µM OSU-2S (MV4-11, U937), for an additional 24 hours. c-Myc overexpression significantly rescued OSU-2S–mediated cytotoxicity (HL-60: n = 9, P = .0041, 86.7% rescue; MV4-11: n = 4, P = .0142, 87.35% rescue, U937: n = 5, P = .0013, 41.9% rescue). Immunoblot analysis (48 hours after transfection) confirmed c-Myc overexpression. (P) Percentage of CD11b⁺ cells in HL-60 or U937 cells transfected with pcDNA3 or pcDNA3-c-Myc and treated 48 hours after transfection with Veh or OSU-2S (1.75 µM for 16-24 hours). c-Myc overexpression significantly rescued OSU-2S–mediated differentiation (HL-60: n = 14, P < .0001, 83.1% rescue; U937: n = 5, P = .0368, 68.9% rescue). (Q) p21 expression in cells transfected with pcDNA3 or pcDNA3-c-Myc and treated 48 hours after transfection with Veh or OSU-2S (1.75 µM [HL-60] or 2.5 µM [MV4-11], 8 hours). Representative western blot images and densitometric quantification of c-Myc (R) and p21 (S), normalized to actin. c-Myc overexpression significantly reversed OSU-2S–mediated p21 upregulation. *P < .05; **P < .01; ***P < .001; ****P < .0001.