Figure 6.

Erythroid cell proliferation and differentiation are impaired in individuals with HEATR3 variants. (A) Bone marrow aspirates of P2 (left panels) and P5 (right panels) with May-Grünwald-Giemsa staining, demonstrating a paucity (arrows) or absence (P5) of erythroid precursors and hypolobulated megakaryocytes (lower panels). (B) Cell count of purified CD34⁺ from the peripheral blood of P2 (orange line) and a healthy control (blue line) subject to the erythroid culture assay was performed (n = 1 to minimize invasiveness). Shown are cell counts on days 5, 7, 10, and 12. (C) Flow cytometry analysis of differentiating erythroid cells from P2 and a healthy control at days 10 and 12. (D) Quantification of panel C. (E) Reverse transcription quantitative real-time polymerase chain reaction quantification of HEATR3 transcript levels in CD34⁺ cells infected with lentiviral constructs expressing shRNAs targeting HEATR3 or a scrambled control (SCR). (F) Cell numbers on days 5, 7, 10, and 12 of an erythroid culture assay after infection with the lentiviral vectors expressing HEATR3 shRNAs. (G) Western blot analysis of the cells on day 7. Blots were probed with antibodies against HEATR3, p21, eS19 (RPS19), heat shock protein 70 (HSP70), GATA1, and pro-caspase 3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. APC, allophycocyanin; EB, early basophilic erythroblasts; LB, late basophilic erythroblasts; ortho, acidophilic erythroblasts; poly, polychromatophilic erythroblasts; proE, proerythroblasts. **P < 0.002; ***P < 0.001; ****P < 0.0001.