Fig. 8. Effect of overexpression of M6P/IGF2R in HL-60R cells on their susceptibility to apoptosis in response to RA. HL-60R cells were transfected with a human M6P/IGF2R cDNA in plasmid pECE or the control pECE vector by the lipofection method. Cells were then incubated with or without 1 µM RA for 72 h, and the expression of M6P/IGF2R, cell growth rate, and cell death were assessed. A, overexpression of M6P/IGF2R was determined by measuring the M6P-inhibitable binding of ß-glucuronidase to saponin-permeabilized cells 48 h after transfection; Control, cells untransfected; Vector, cells transfected with pECE; M6P/IGF2R, cells transfected with M6P/IGF2R cDNA. B, cell growth rate, as measured by counting the viable cells of each treatments; , vector-transfected cells without RA treatment; , vector-transfected cells with RA treatment; , M6P/IGF2R cDNA-transfected cells without RA treatment; , M6P/IGF2R cDNA-transfected cells with RA treatment. C, cytospin analysis of apo-ptosis. Cells transfected with the control vector (a and b) or with M6P/IGF2R cDNA (c and d) were treated with (b and d) or without (a and c) 1 µM RA for 3 days, centrifuged onto slides, and stained with LeukoStat stain. Arrows, typical apoptotic cells with pyknotic or fragmented nuclei.D, flow cytometry analysis of apoptosis. Cells transfected with control vector (a and b) or with M6P/IGF2R cDNA (c and d) were treated with (b and d) or without (a and c) 1 µM RA for 3 days and stained with FITC-labeled annexin V. The cells (10,000) were then analyzed by flow cytometry. The percentage of cells stained by FITC-labeled annexin V in each sample is indicated.