Forced expression of NELF-E inhibits granulocytic differentiation. (A) Mouse 32Dcl3 cells were stably transfected with empty vector (ctrl) or Flag-tagged NELF-E (Flag-NE) and blotted with anti-Flag antibody. (B) Western blot of NELF-A and -E protein from 32D cells transfected with or without Flag-tagged NELF-E (Flag-NE). Protein extraction was done in undifferentiated cells (day 0) and cells differentiated for 1 day (d1) and 4 days (d4). Two independent Flag-NE transfected cell lines were shown. Red asterisk indicates Flag-NE that is slightly bigger than endogenous NELF-E. GAPDH is used as a loading control. (C) Representative images of MGG staining showing the morphology of human granulocytic differentiation from myeloblasts to mature segmented neutrophils. (D) MGG staining to compare the morphology of murine 32D cells expressing Flag-NE with control cells during neutrophil differentiation (original magnification ×200). (E) MGG staining to compare morphology of differentiating cells from human CD34⁺ HSPCs treated with dimethyl sulfoxide (DMSO) vs flavopiridol (Flavo) (original magnification ×200). (D-E) The percentages of different cell types at various time points during differentiation are shown on the bar charts. Quantitative results represent average percentages by counting 100 cells per experiment from 3 independent experiments.