Analysis of differentiation markers in untreated M1, M1myc, M1Egr-1, and M1Egr-1/myc6 cells. (A) Lysozyme expression was determined by Northern blot analysis. Total RNA was extracted from 0.8 × 10⁶ cells, loaded on formaldehyde-agarose gels (10 mg/lane), and transferred to Duralon nylon membranes for analysis, as described in “Materials and methods.” Blots were hybridized with ³²P-labeled lysozyme cDNA probe (see “Materials and methods”). This is a representative experiment that was carried out 3 times. (B) F4-80 macrophage-specific antigen expression was assayed by FACS analysis. Cells (2.0 × 10⁶) were incubated with a fluorescein isothiocyanate (FITC)–linked anti–F4-80 antibody, and fluorescence was measured using flow cytometry, as described in “Materials and methods.” Results are the average of 3 experiments, with an SD of up to 15%. The differences between M1 and M1myc were P > .05 (not significant), between M1 and M1Egr-1 P < .01 (significant), between M1 and M1Egr-1/myc P < .05 (significant), and between M1Egr-1 and M1Egr-1/myc P < .05 (significant). (C) Morphologic characteristics of cells were determined counting at least 300 cells from cytospin smears stained with May-Grünwald-Giemsa, scoring the proportion of immature blasts, cells at intermediate stages of differentiation, and mature macrophages (see “Materials and methods”). Results are the average of 3 experiments, with an SD for each cell clone and morphologic type of up to 10%. Using the intermediate stage of differentiation as a measure of differentiation of the untreated population of cells, the differences between M1 and M1myc were P > .1 (not significant), between M1 and M1Egr-1 P < .01 (significant), between M1 and M1Egr-1/myc P < .05 (significant), and between M1Egr-1 and M1Egr-1/myc P < .05 (significant).