Figure 3.
PP2A–mediated leukemic maturation. (A) Increase in the percentage of CD11b+/CD14+ or CD11b+ cells (CD11b-FITC or CD11b-BV421, CD14-BV650) in the HL-60 (n = 6, P = .001) and U937 cell lines (n = 5, P = .0011) treated with OSU-2S (2.5 μM, 48 hours) compared with vehicle control (Veh). (B) Increased percentage of CD33+/CD11b+ (CD33-PE, CD11b-FITC, or CD11b-BV421) primary AML cells (n = 8; AML14-16, 20, 24-27) treated with OSU-2S (3.5 μM, 48 hours) compared with Veh (P = .0384; 19.93% mean increase in CD11b+ cells). (C) Representative morphological changes in HL-60 and primary AML cells (AML14) treated with OSU-2S. (D) Percentage of CD11b+ cells transfected with negative control (nc) siRNA or PP2Ac siRNA and treated 24 hours after transfection with Veh or OSU-2S (1.75 µM) for an additional 16 to 24 hours. OSU-2S–mediated induction of CD11b was significantly reversed by siRNA knockdown of PP2Ac (HL-60: n = 7, P < .0001, 66.64% rescue; U937: n = 6, P = .0004, 89.69% rescue). (E) Percentage of CD11b+ HL-60 cells transfected with either nc siRNA or B56α siRNA and treated 24 hours after transfection with Veh or OSU-2S (1.75 µM) for an additional 16 to 24 hours. OSU-2S–mediated induction of CD11b was significantly reversed by siRNA knockdown of PP2A B56α (n = 12, P = .017, 67.7% rescue). (F) Increase in percentage of CD11b+ cells with 2.5 µM FTY720 in HL-60 (48 hours; n = 5; P = .0338) and U937 cells (72 hours; n = 7, P = 0016) compared with Veh. (G) Increase in percentage of CD11b+ cells with 10 µM PPZ in HL-60 (48 hours; n = 5, P = .0002) and U937 cells (72 hours; n = 7, P = .0012) compared with Veh. (H) Increase in percentage of CD11b+ cells in primary AML cells (n = 5; AML 15, 18, 24, 38, and 44) treated with FTY720 (2.5 µM, 48 hours, P = .0375, 7.054% increase) or PPZ (10 µM, 48 hours, P = .0181, 5.668% increase) compared with Veh. (I) Representative images of mass cytometric analysis of primary AML cells treated with Veh or OSU-2S (3 μM, 48 hours) revealed increased surface differentiation markers CD14 and CD15. (J) Mass cytometric analysis of HL-60 cells treated with Veh or OSU-2S revealed an increase in the surface differentiation markers CD14, CD15, and CD66b. *P < .05; **P < .01; ***P < .001; ****P < .0001.

PP2A–mediated leukemic maturation. (A) Increase in the percentage of CD11b+/CD14+ or CD11b+ cells (CD11b-FITC or CD11b-BV421, CD14-BV650) in the HL-60 (n = 6, P = .001) and U937 cell lines (n = 5, P = .0011) treated with OSU-2S (2.5 μM, 48 hours) compared with vehicle control (Veh). (B) Increased percentage of CD33+/CD11b+ (CD33-PE, CD11b-FITC, or CD11b-BV421) primary AML cells (n = 8; AML14-16, 20, 24-27) treated with OSU-2S (3.5 μM, 48 hours) compared with Veh (P = .0384; 19.93% mean increase in CD11b+ cells). (C) Representative morphological changes in HL-60 and primary AML cells (AML14) treated with OSU-2S. (D) Percentage of CD11b+ cells transfected with negative control (nc) siRNA or PP2Ac siRNA and treated 24 hours after transfection with Veh or OSU-2S (1.75 µM) for an additional 16 to 24 hours. OSU-2S–mediated induction of CD11b was significantly reversed by siRNA knockdown of PP2Ac (HL-60: n = 7, P < .0001, 66.64% rescue; U937: n = 6, P = .0004, 89.69% rescue). (E) Percentage of CD11b+ HL-60 cells transfected with either nc siRNA or B56α siRNA and treated 24 hours after transfection with Veh or OSU-2S (1.75 µM) for an additional 16 to 24 hours. OSU-2S–mediated induction of CD11b was significantly reversed by siRNA knockdown of PP2A B56α (n = 12, P = .017, 67.7% rescue). (F) Increase in percentage of CD11b+ cells with 2.5 µM FTY720 in HL-60 (48 hours; n = 5; P = .0338) and U937 cells (72 hours; n = 7, P = 0016) compared with Veh. (G) Increase in percentage of CD11b+ cells with 10 µM PPZ in HL-60 (48 hours; n = 5, P = .0002) and U937 cells (72 hours; n = 7, P = .0012) compared with Veh. (H) Increase in percentage of CD11b+ cells in primary AML cells (n = 5; AML 15, 18, 24, 38, and 44) treated with FTY720 (2.5 µM, 48 hours, P = .0375, 7.054% increase) or PPZ (10 µM, 48 hours, P = .0181, 5.668% increase) compared with Veh. (I) Representative images of mass cytometric analysis of primary AML cells treated with Veh or OSU-2S (3 μM, 48 hours) revealed increased surface differentiation markers CD14 and CD15. (J) Mass cytometric analysis of HL-60 cells treated with Veh or OSU-2S revealed an increase in the surface differentiation markers CD14, CD15, and CD66b. *P < .05; **P < .01; ***P < .001; ****P < .0001.

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