ΔP1 v-Myb cells express erythroid specific genes.
(A) Growth kinetics of chicken blastoderm cells infected with NeoAMVwt (wt) and NeoAMVΔP1 (ΔP1) viruses are shown. (B) Cytochemical staining of wild-type and ΔP1 v-Myb cells on days 7, 15, and 30 of culture. Benzidine staining reveals hemoglobinized erythroid cells (yellow-brown); cells were counterstained with Diff-Quik to show monoblasts (light blue). The bar indicates 15 μm.(C) v-Myb proteins in 106 wild-type and ΔP1 v-Myb cells (day 20) were analyzed by western blotting using the myb-specific monoclonal antibody. Equal amounts of proteins in loaded samples were verified by Ponceau staining of the western blot. v-Myb synthesized in the baculovirus expression system (Bac) and in 106 cells of the v-myb–transformed BM2 cell line (BM2) are shown as controls. (D) Flow cytometry of wild-type and ΔP1 v-Myb cells on days 7 and 15 using monoclonal antibodies MC51/2 (blue) for myeloid and JS4 (red) for erythroid surface antigens. Empty curves represent negative controls. Intensity of fluorescence is plotted on horizontal axes, amounts of measured cells on vertical axes. (E) mRNAs for c-Myb, GATA-1, PU.1, Egr-1, and multiple SCL mRNAs in wild-type and ΔP1 v-Myb cells (day 15) were analyzed by northern blotting; C/EBPβ and GBX2 mRNAs were analyzed by RNase protection. RNAs of myeloid BM2 and erythroid HD4 cell lines were analyzed for comparison. Actin and GAPDH probes were used as controls of equal loading in RNase protection and northern blot assays, respectively. Two independent experiments provided identical results.
