Influence of nucleophosmin-1 (NPM-1) on erlotinib-mediated cytolysis. (A) Typical immunofluorescence images obtained after staining of KG-1 cells with antibodies specific for NPM-1 and p14^ARF. Chromatin was counterstained using DAPI. Note the nuclear localization of NPM-1 and p14^ARF that is lost after erlotinib treatment (10 μM, 30 minutes) in favor of a cytoplasmic staining. (B) Quantitation of the data obtained as in panel A, 30 minutes or 3 hours after addition of erlotinib. (C-F) Failure of erlotinib to affect the subcellular localization of NPM-1 (C-F), p14^ARF (C,D) and NF-κB p65 (E,F) in erlotinib-resistant P39 cells. Representative immunofluorescence microphotographs are shown in panels C and E, and quantitative results are reported in panels D and F. (G-I) Effect of NPM-1 knockdown on the apoptosis-inducing capacity of erlotinib in P39 and KG-1 cells. NPM-1 was knocked down with 2 distinct siRNA heteroduplexes (insets in panels H and I). Twenty-four hours after transfection erlotinib (or DMSO as a solvent control) was added for 48 hours, and the frequency of dying and dead cells was measured using the annexin V/PI method. Representative FACS pictograms are shown for P39 cells in panel G, and quantitative results (X ± SD, n = 3) are depicted for both P39 and KG-1 cells in panels H and I.