Characteristics of APL blasts.

(A) Mice showing overt signs of disease were killed, and spleen cells were analyzed for expression of several surface markers. Spleen cells from untreated mice are shown for comparison in the left panels. PML-RAR blasts are CD34⁺/C-KIT⁺/MAC1⁺/GR1⁺. (B,C) In panel B, expression of PML-RAR in APL blasts was confirmed (Bi) by analysis of PML-RAR expression by immunoprecipitation with an anti-PML antibody, followed by Western blot using anti-RAR antibodies. Spleen cell extracts were prepared from control and leukemic animals obtained as outlined in Figure 1, or leukemic animals derived from cathepsin G-PML-RAR transgenic mice.8 Panel Bii shows RT-PCR analysis of RNA obtained from the bone marrow of 3 independently derived leukemic mice (L1-L3). Reverse transcriptase was omitted in the samples marked with the minus sign as control. Negative control (lanes “C”) is from a control mouse, positive control (lanes “PR9”) derives from U937 cells stably transfected with PML-RAR; the arrow indicates the specific, amplified PCR product. (C) Immunofluorescence of bone marrow cells from a leukemic mouse prior to (Cii), or after 5 days of RA treatment (Ciii), using antibodies against mouse PML (original magnification × 630). Control, normal bone marrow cells (Ci).