Journal: Journal of Sickle Cell Disease

Article Title: Hydroxyurea reduces the levels of the fetal globin gene repressors ZBTB7A/LRF and BCL11A in erythroid cells in vitro

doi: 10.1093/jscdis/yoae008

Figure Lengend Snippet: Hydroxyurea (HU) treatment of murine erythroleukemia (MEL) cells induces mouse embryonic/fetal β-like globins and reduces the levels of their transcriptional repressors ZBTB7A and BCL11A. (A) Mouse embryonic/fetal (ε y and β h1 ) and adult (β maj and β min ) β-like globin mRNA expression in MEL cells treated with HU. The mean ± SD is shown, with dots representing three experimental replicates for either vehicle-treated (–HU) or 100 μM hydroxyurea (+HU) treatment conditions. The globin percentages are shown as a proportion of the total β-like globins (sum of ε y , β h1 , β maj , and β min ). (B) Expression of Zbtb7a and Bcl11a mRNA in vehicle-treated (–HU) or hydroxyurea (+HU)-treated MEL cells. The mean ± SD is shown, with dots representing three experimental replicates. Bcl11a mRNA expression includes the XL, L, and S splice forms. (C and D) Western blot for ZBTB7A (C) and BCL11A-XL (D) proteins in vehicle-treated (–) or 100 μM HU (+)-treated MEL cells. Three independent experimental replicates are shown with β-ACTIN as a loading control. A MEL cell line where the entire BCL11A gene has been deleted via CRISPR-Cas9-mediated genome editing is included as a control and size standard (BCL11A KO). (E) Densitometry analysis for the ZBTB7A and BCL11A-XL Western blots shown in (C) and (D) with each symbol representing a separate experiment. Shown is the mean ± SD. Significance determined by an unpaired t -test is indicated as P > .05 (ns), P ≤ .05 (*), P ≤ .01 (**), P ≤ .0001 (****).

Article Snippet: Additionally, CD34 + cells were harvested on day 10 for Western blot analysis of BCL11A (ab19487, abcam), ZBTB7A (sc-33683, Santa Cruz Biotechnology Inc), and GATA1 (ab11852, abcam) protein levels.

Techniques: Expressing, Western Blot, Control, CRISPR

Journal: Journal of Sickle Cell Disease

Article Title: Hydroxyurea reduces the levels of the fetal globin gene repressors ZBTB7A/LRF and BCL11A in erythroid cells in vitro

doi: 10.1093/jscdis/yoae008

Figure Lengend Snippet: Treatment of human CD34 + cell-derived erythroblasts with hydroxyurea induces fetal globin s and reduces the levels of fetal globin repressors, ZBTB7A and BCL11A, and GATA1. Normal donor CD34 + hematopoietic stem and progenitor cells (HSPCs) ( n = 3 independent donors) were induced to undergo erythroid differentiation on day 1. Hydroxyurea (HU) was added at the indicated concentrations (0-40 μM), beginning on either day 1, 3, or 8. (A) Fetal hemoglobin (HbF) protein levels determined by ion-exchange high-performance liquid chromatography (HPLC) on day 14. HbF is indicated as the proportion of fetal and adult hemoglobin (HbF/(HbF + HbA)). (B) HU dose-response measurements during the course of erythroid differentiation, with HU added on day 1 (open circle) or 3 (shaded square). Cells were analyzed for mRNA or protein in differentiation days 7 and 10, respectively, with erythroid differentiation assessed by flow cytometry on days 8 and 14 for maturation markers CD235a + and CD49d + /Band3 + (as a percentage of CD235a + cells). (C) F-cell levels were analyzed on day 14 by flow cytometry using an anti-HbF antibody. (D) γ-globin mRNA levels determined on day 7. The percentage of γ-globin ( HBG1/2 ) is shown as a proportion of total β-like globins (γ /γ + β). (E) mRNA expression of ZBTB7A , BCL11A (XL, L, and S splice forms), KLF1 and GATA1 was determined on day 7. (F) Western blots for ZBTB7A (top), BCL11A-XL (middle), and GATA1 (bottom) determined on day 10. Days 1 and 3 indicate the day HU was added to the erythroid differentiation. Representative data from 1 CD34 + donor are shown. shows the results of Western blots from 3 biological replicate experiments using 3 different CD34+ cell donors. β-ACTIN or Histone H3 are shown as loading controls. (G) Densitometry analysis for the ZBTB7A, BCL11A-XL, and GATA1 Western blots shown in (F). The mean ± SD are shown. Asterisks represent significance from a two-way ANOVA (relative to 0 μM HU) with Tukey’s multiple comparison testing. Black and grey asterisks represent significant differences between controls (0 μM) and HU added at either day 1 or day 3, respectively: (*) P ≤ .05, (**) P ≤ .01, (***) P ≤ .001, (****) P ≤ .0001.

Article Snippet: Additionally, CD34 + cells were harvested on day 10 for Western blot analysis of BCL11A (ab19487, abcam), ZBTB7A (sc-33683, Santa Cruz Biotechnology Inc), and GATA1 (ab11852, abcam) protein levels.

Techniques: Derivative Assay, High Performance Liquid Chromatography, Flow Cytometry, Expressing, Western Blot, Comparison