Figure 3. Morphological and functional assessment of the differentiation-inducing activity of EGFR inhibitors. (A–F). Human acute myeloid leukemia HL-60 cells were treated with 10 μM erlotinib (ERLO), 10 μM gefitinib (GEFI), or an equal volume of DMSO, alone or in combination with 100 nM all-trans retinoic acid (ATRA), or 50 nM 1α,25-hydroxycholecalciferol (VD), for 3 d, then cytospun and processed for the cytochemical assessment of myeloid differentiation upon May–Grünwald–Giemsa staining (Aand B) or the colorimetric detection of NADPH oxidase (C and D) or α-naphtyl acetate esterase (E and F) enzymatic activities. Panels (A, C, and E) depict representative images (scale bars = 10 µm), whereas quantitative data on the percentage of cells exhibiting morphological signs of differentiation (decreased cytoplasmic basophilia, nuclear lobulation and cytoplasmic granulation), nitroblue tetrazolium chloride (NBT)-reducing activity and α-naphtyl acetate esterase activity are reported in (B, D, and F), respectively (means ± SEM; n = 3 with at least 100 cells/condition). The inset in (E) depicts a megakaryocyte (M) exhibiting intense α-naphtyl acetate esterase activity (positive control). *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA plus Dunnett post-hoc test), as compared with DMSO-treated cells; n.s., not significant, ###P < 0.001 (ANOVA plus Bonferroni post-hoc test), as compared with ATRA-treated cells; ++P < 0.01, +++P < 0.001 (ANOVA plus Bonferroni post-hoc test), as compared with VD-treated cells.