American Society of Hematology

Figure 3.

$p53 is activated during erythroid differentiation. (A) Human primary erythroblasts were derived from CD34+ progenitors cultured with SCF, IL-6, and IL-3 for 6 days (with the addition of dexamethasone at day 1); with SCF and EPO between days 7 and 11; and then with EPO alone. (B) Proportion of progenitors, ProEs, baso1s, baso2s, polyEs, and orthoEs by cytological examination of May-Grünwald-Giemsa–stained cytospins. Mean ± SEM of 3 independent experiments. (C) Expression of the SCF receptor CD117 and GlyA by flow cytometry during human erythroblast differentiation. p53 expression in human primary erythroblasts at different days in culture by immunofluorescence imaging of p53 and 4′,6-diamidino-2-phenylindole (DAPI) staining, with merged images of the 2 stains. Images were obtained on a Leica DMI6000 inverted microscope with spinning disk and analyzed with ImageJ. Original magnification ×100. (D) Western blot analysis of phospho-p53 serine 15 (pp53ser15), p53, and p53 target p21/Cdkn1A during normal erythroblast differentiation. Hsc70 was used as the loading control. (E) Western blot analysis of p53 in nuclear and cytosolic fractions. GATA1 was used as the loading control for the nuclear fraction (n), and the p85 subunit of phosphatidylinositol 3 kinase was used as the loading control for the cytosolic fraction (c). (F) Quantification of nuclear and cytoplasmic expression of p53 is expressed as the nuclear/cytoplasmic ratio. (G) Timeline of GFP-shRNA lentivirus transduction at days 8 and 9 and control of GFP expression at day 10. Cells were analyzed during the 4 days after the last transduction (day 13). (H) Western blot analysis of TP53 knockdown by shRNA at day 13. An SCR shRNA was the control. pp53ser15, p53, and p21/Cdkn1A protein expression are shown. Actin was used as the loading control. (H) Erythroid differentiation was assessed by the quantification of GlyA expression by flow cytometry. Results are expressed as mean percentages ± SEM of 3 experiments. (I) qRT-PCR measurement of p53 target genes quantities in shTP53 and shSCR erythroblasts. Transcript levels were normalized to B2M, UBC, and ACTB levels. Results are expressed as mean NRQs ± SEM of 3 experiments. (J) Proliferation curve of shTP53 and shSCR erythroblasts in a cumulative number of cells. (K) GlyA expression in shTP53 and shSCR erythroblasts. Mean percentages ± SEM of 3 experiments. (L) Proportions of erythroblasts by cytological examination of May-Grünwald Giemsa–stained cytospins at day 13. Mean percentage ± SEM of 3 experiments. (M) Apoptosis measured as the mean percentage ± SEM of annexin V+ cells in 3 experiments. (N) Design of mouse experiments. FVB/N Tp53−/− or Tp53wt mice were treated with 40 mg/kg CX-5461 for 24 hours. (O-P) Bone marrows were collected and the percentage of erythroid nucleated cells (O) and the absolute number of proE, baso, poly, ortho, and reticulocytes (P) were determined by flow cytometry. *P < .05; **P < .01; ***P< .001, by Student t test.$

p53 is activated during erythroid differentiation. (A) Human primary erythroblasts were derived from CD34⁺ progenitors cultured with SCF, IL-6, and IL-3 for 6 days (with the addition of dexamethasone at day 1); with SCF and EPO between days 7 and 11; and then with EPO alone. (B) Proportion of progenitors, ProEs, baso1s, baso2s, polyEs, and orthoEs by cytological examination of May-Grünwald-Giemsa–stained cytospins. Mean ± SEM of 3 independent experiments. (C) Expression of the SCF receptor CD117 and GlyA by flow cytometry during human erythroblast differentiation. p53 expression in human primary erythroblasts at different days in culture by immunofluorescence imaging of p53 and 4′,6-diamidino-2-phenylindole (DAPI) staining, with merged images of the 2 stains. Images were obtained on a Leica DMI6000 inverted microscope with spinning disk and analyzed with ImageJ. Original magnification ×100. (D) Western blot analysis of phospho-p53 serine 15 (pp53^ser15), p53, and p53 target p21/Cdkn1A during normal erythroblast differentiation. Hsc70 was used as the loading control. (E) Western blot analysis of p53 in nuclear and cytosolic fractions. GATA1 was used as the loading control for the nuclear fraction (n), and the p85 subunit of phosphatidylinositol 3 kinase was used as the loading control for the cytosolic fraction (c). (F) Quantification of nuclear and cytoplasmic expression of p53 is expressed as the nuclear/cytoplasmic ratio. (G) Timeline of GFP-shRNA lentivirus transduction at days 8 and 9 and control of GFP expression at day 10. Cells were analyzed during the 4 days after the last transduction (day 13). (H) Western blot analysis of TP53 knockdown by shRNA at day 13. An SCR shRNA was the control. pp53^ser15, p53, and p21/Cdkn1A protein expression are shown. Actin was used as the loading control. (H) Erythroid differentiation was assessed by the quantification of GlyA expression by flow cytometry. Results are expressed as mean percentages ± SEM of 3 experiments. (I) qRT-PCR measurement of p53 target genes quantities in shTP53 and shSCR erythroblasts. Transcript levels were normalized to B2M, UBC, and ACTB levels. Results are expressed as mean NRQs ± SEM of 3 experiments. (J) Proliferation curve of shTP53 and shSCR erythroblasts in a cumulative number of cells. (K) GlyA expression in shTP53 and shSCR erythroblasts. Mean percentages ± SEM of 3 experiments. (L) Proportions of erythroblasts by cytological examination of May-Grünwald Giemsa–stained cytospins at day 13. Mean percentage ± SEM of 3 experiments. (M) Apoptosis measured as the mean percentage ± SEM of annexin V⁺ cells in 3 experiments. (N) Design of mouse experiments. FVB/N Tp53^−/− or Tp53^wt mice were treated with 40 mg/kg CX-5461 for 24 hours. (O-P) Bone marrows were collected and the percentage of erythroid nucleated cells (O) and the absolute number of proE, baso, poly, ortho, and reticulocytes (P) were determined by flow cytometry. *P < .05; **P < .01; ***P< .001, by Student t test.

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