Figure 2
Figure 2. PRAME expression in all-trans retinoic acid–responsive leukemia cell lines increases proliferation and inhibits granulocytic differentiation. (A) PRAME protein expression is shown in HL60, NB4, and K562 cells by Western blotting. PRAME protein overexpression by lentiviral PRAME expression vector transduction is shown in total cell lysates (T) from HL60 cells and in both cytoplasmic (C) and nuclear (N) lysates from NB4 cells. PRAME silencing using lentiviral vectors containing short-hairpins targeting PRAME is shown in T-cell lysates in HL60 cells and N lysates in K562 cells. In the HL60 cells shown here, PRAME was silenced in HL60 cells overexpressing PRAME protein. Lamin served as the nuclear control; beta actin and GAPDH served as the cytoplasmic or total lysate control. (B) PRAME-overexpressing HL60 cells (PRAME) proliferated more rapidly than control cells (Control) over 96 hours. When PRAME was silenced in these PRAME-overexpressing cells (shPRAME), proliferation decreased compared with control cells (shControl). PRAME cells are PRAME-overexpressing cells; control cells are the empty vector control. PRAME was silenced (shPRAME) in both PRAME-overexpressing cells and the corresponding empty vector–transduced cells. shControl is a hairpin that targets GFP. (C) The increase in proliferation in HL60 PRAME cells relative to control cells (P < .001) as well as the decrease in proliferation of shPRAME HL60 cells relative to shControl HL60 cells (P = .06 in PRAME-overexpressing cells and P < .001 in control HL60 cells) were seen both in the absence and presence of increasing concentrations of all-trans retinoic acid (ATRA). For simplicity, proliferation in PRAME or shPRAME cells is shown relative to the corresponding control cell line (experimental condition/control condition). Thus, the line at 1.00 indicates the expected ratio if there is no difference. Data at 96 hours are used and show the mean of 3 independent experiments. The P values, however, were calculated using all data (ATRA and no ATRA) at 0, 24, 48, 72, and 96 hours. (D) Granulocytic differentiation as measured by CD11b expression over time was inhibited in PRAME-overexpressing HL60 cells. PRAME cells expressed significantly less CD11b over time than control cells at various ATRA concentrations (P = .003). When PRAME was silenced in these PRAME-overexpressing cells as well as the empty vector control cells, CD11b expression was greater in shPRAME cells compared with shControl cells in both the PRAME-overexpressing cell line and the control vector cell line (P < .001 for both). The ratio of CD11b expression in PRAME or shPRAME cells is shown relative to CD11b expression in control or shControl cells (experimental percentages CD11b/control percentage CD11b) after 96 hours. The line at 1.00 indicates the expected ratio if there is no difference.

PRAME expression in all-trans retinoic acid–responsive leukemia cell lines increases proliferation and inhibits granulocytic differentiation. (A) PRAME protein expression is shown in HL60, NB4, and K562 cells by Western blotting. PRAME protein overexpression by lentiviral PRAME expression vector transduction is shown in total cell lysates (T) from HL60 cells and in both cytoplasmic (C) and nuclear (N) lysates from NB4 cells. PRAME silencing using lentiviral vectors containing short-hairpins targeting PRAME is shown in T-cell lysates in HL60 cells and N lysates in K562 cells. In the HL60 cells shown here, PRAME was silenced in HL60 cells overexpressing PRAME protein. Lamin served as the nuclear control; beta actin and GAPDH served as the cytoplasmic or total lysate control. (B) PRAME-overexpressing HL60 cells (PRAME) proliferated more rapidly than control cells (Control) over 96 hours. When PRAME was silenced in these PRAME-overexpressing cells (shPRAME), proliferation decreased compared with control cells (shControl). PRAME cells are PRAME-overexpressing cells; control cells are the empty vector control. PRAME was silenced (shPRAME) in both PRAME-overexpressing cells and the corresponding empty vector–transduced cells. shControl is a hairpin that targets GFP. (C) The increase in proliferation in HL60 PRAME cells relative to control cells (P < .001) as well as the decrease in proliferation of shPRAME HL60 cells relative to shControl HL60 cells (P = .06 in PRAME-overexpressing cells and P < .001 in control HL60 cells) were seen both in the absence and presence of increasing concentrations of all-trans retinoic acid (ATRA). For simplicity, proliferation in PRAME or shPRAME cells is shown relative to the corresponding control cell line (experimental condition/control condition). Thus, the line at 1.00 indicates the expected ratio if there is no difference. Data at 96 hours are used and show the mean of 3 independent experiments. The P values, however, were calculated using all data (ATRA and no ATRA) at 0, 24, 48, 72, and 96 hours. (D) Granulocytic differentiation as measured by CD11b expression over time was inhibited in PRAME-overexpressing HL60 cells. PRAME cells expressed significantly less CD11b over time than control cells at various ATRA concentrations (P = .003). When PRAME was silenced in these PRAME-overexpressing cells as well as the empty vector control cells, CD11b expression was greater in shPRAME cells compared with shControl cells in both the PRAME-overexpressing cell line and the control vector cell line (P < .001 for both). The ratio of CD11b expression in PRAME or shPRAME cells is shown relative to CD11b expression in control or shControl cells (experimental percentages CD11b/control percentage CD11b) after 96 hours. The line at 1.00 indicates the expected ratio if there is no difference.

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