Pml^C62A/C65Aexpression induces mislocalization of NB constituents, Pml SUMOylation deficiency, and expansion of the LSK compartment. (A) Representative confocal microscopy images are presented for Pml (green), Daxx, Cbp, and Sumo-1 (red) staining in Pml^WT, Pml^C62A/C65A, and Pml^−/− MEFs. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 10 μm. (B) Expression levels demonstrated by western blot analysis of whole-cell lysates extracted from Pml^WT, Pml^C62A/C65A, and Pml^−/− MEFs. The asterisk (*) indicates a nonspecific band. Tubulin was used as loading control. (C) Representative images of DAXX (red) and PML (green) staining in NB4 cells treated with DMSO (vehicle control) or ATRA (1 μM). Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. (D) MEFs were treated with or without ATO (1 μM) for 1 hour, as indicated, followed by immunoprecipitation (IP) by control immunoglobulin G (IgG; CTRL) or with antibodies against the indicated protein. The immunoprecipitates were analyzed by immunoblot (IB) with an anti-Pml antibody in order to reveal the SUMOylated or ubiquitinated forms of Pml. Data shown are representative of 3 independent experiments. (E) Relative number of LSK cells in BM at 8 weeks (n ≥ 3). (F) Cell-cycle phase distribution of LSK cells labeled with Click-iT EdU in vivo (n ≥ 3). (G) Representative dot plots of cell-cycle status of LSK population. (E-F) Two-tailed unpaired Student t test analyses were performed.