Figure 3
Figure 3. NPMc protects against dichotomous effects of TRAIL treatment in HL-60 and Ara-C treatment in CD34+ primary blood cells. (A) Top: Apoptosis scored with condensed nuclei in Hoechst-stained HL-60 cells transfected with GFP or GFP-tagged NPMc and exposed to 100 ng/mL TRAIL for up to 48 hours. Bottom: Representative micrographs of HL-60 cells transfected with GFP and GFP-NPMc, with insets of micrographs at higher magnifications, and of nuclear staining with Hoechst 33342 showing condensed nuclei (white arrowheads). Unless otherwise stated, scale bars represent 10 μm. (B) Top: Number of cells with differentiation beyond the promyeloid stage was scored in May-Grünwald-Giemsa–stained HL-60 cells transfected with GFP or GFP-tagged NPMc, with or without 100 ng/mL TRAIL treatment. At least 150 cells were scored for each set of experiments. Bottom: Representative micrographs of May-Grünwald-Giemsa staining of TRAIL-treated GFP- or GFP-tagged NPMc-transfected cells. Black arrows represent differentiated cells; scale bar represents 15 μm. (C) Top: Confocal microscopy image of primary blood cells showing entire cell and cytoplasmic localization of GFP and GFP-tagged NPMc, respectively. Middle: Apoptosis scored with condensed nuclei in Hoechst-stained cord blood-derived CD34+ cells transduced with GFP or GFP-tagged NPMc and exposed to 5μM Ara-C for up to 48 hours. Bottom: Representative micrographs of nuclear staining of CD34+ cells showing condensed nuclei (white arrowheads). Scale bars represent 10 μm. (D) Flow cytometric analysis of GFP or GFP-tagged NPMc-transduced CD34+ cells. Top: GFP+ cells were gated and analyzed for immunostaining of differentiation markers CD11b and CD14 before transduction (day −2), 2 days after transduction (day 0), and days 2, 4, and 7 days after stimulation with 100 U/mL IL-3 (days 2, 4, and 7). (E) Cleaved caspase-6 and -8 were pulled down with glutathione-Sepharose–immobilized GST or GST-NPM from cytosolic extracts from HL-60 cells that were apoptotically challenged with cytochrome c and dATP as in Figure 1A. (F) Cells from panel A were lysed, and caspase-6 and -8 activities were measured using pNA-tagged caspase substrates. **P < .01. Data were obtained from 3 sets of experiments and presented as mean ± SEM.

NPMc protects against dichotomous effects of TRAIL treatment in HL-60 and Ara-C treatment in CD34+ primary blood cells. (A) Top: Apoptosis scored with condensed nuclei in Hoechst-stained HL-60 cells transfected with GFP or GFP-tagged NPMc and exposed to 100 ng/mL TRAIL for up to 48 hours. Bottom: Representative micrographs of HL-60 cells transfected with GFP and GFP-NPMc, with insets of micrographs at higher magnifications, and of nuclear staining with Hoechst 33342 showing condensed nuclei (white arrowheads). Unless otherwise stated, scale bars represent 10 μm. (B) Top: Number of cells with differentiation beyond the promyeloid stage was scored in May-Grünwald-Giemsa–stained HL-60 cells transfected with GFP or GFP-tagged NPMc, with or without 100 ng/mL TRAIL treatment. At least 150 cells were scored for each set of experiments. Bottom: Representative micrographs of May-Grünwald-Giemsa staining of TRAIL-treated GFP- or GFP-tagged NPMc-transfected cells. Black arrows represent differentiated cells; scale bar represents 15 μm. (C) Top: Confocal microscopy image of primary blood cells showing entire cell and cytoplasmic localization of GFP and GFP-tagged NPMc, respectively. Middle: Apoptosis scored with condensed nuclei in Hoechst-stained cord blood-derived CD34+ cells transduced with GFP or GFP-tagged NPMc and exposed to 5μM Ara-C for up to 48 hours. Bottom: Representative micrographs of nuclear staining of CD34+ cells showing condensed nuclei (white arrowheads). Scale bars represent 10 μm. (D) Flow cytometric analysis of GFP or GFP-tagged NPMc-transduced CD34+ cells. Top: GFP+ cells were gated and analyzed for immunostaining of differentiation markers CD11b and CD14 before transduction (day −2), 2 days after transduction (day 0), and days 2, 4, and 7 days after stimulation with 100 U/mL IL-3 (days 2, 4, and 7). (E) Cleaved caspase-6 and -8 were pulled down with glutathione-Sepharose–immobilized GST or GST-NPM from cytosolic extracts from HL-60 cells that were apoptotically challenged with cytochrome c and dATP as in Figure 1A. (F) Cells from panel A were lysed, and caspase-6 and -8 activities were measured using pNA-tagged caspase substrates. **P < .01. Data were obtained from 3 sets of experiments and presented as mean ± SEM.

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