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HbF rescues dyserythropoiesis in SCD
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sis. This inference was supported by our findings showing lower percentages of low F-cells in SCD cultures as com- pared to control, as well as lower percentages of SCD low F-cells under hypoxic than normoxic conditions (Figure 3H). On the other hand, the percentage of high F-cells was significantly higher in SCD erythroblasts as compared to control, with even higher percentages under hypoxia (Figure 3I). Of note, no difference in the percentage of low or high F-cells was observed between hypoxia and nor- moxia for control cells (Figure 3H and I), indicating that the differences observed for SCD cells are the result of differ- ential cell death related to HbF expression levels.
HbS-HSP70 protein complex in sickle cell disease cells under hypoxia
In order to explore the molecular mechanism of cyto- plasmic trapping of HSP70, we performed immunofluores- cence assays using confocal microscopy and found co- localization of HSP70 and hemoglobin in both control and SCD cells (Figure 4A). We used Pearson’s correlation coef- ficient23,24 to assess the degree of co-localization and found no difference between both cell types (data not shown), probably because of the high abundance of hemoglobin in the cytoplasm. In order to overcome this limitation, we performed PLA, that show fluorescent spots when two proteins are at a distance <40 nm.25 We detected SCD cells with fluorescent spots (PLA+) under both normoxic and hypoxic conditions (Figure 4B) that were further quantified by imaging flow cytometry (Figure 4C). The percentage of PLA+ cells was very low in control, with values close to background levels (1-1.5%) under both normoxia and hypoxia, while SCD cells showed higher values under nor- moxia (4.5-18.5%) with a systematic and significant increase under hypoxia (9.5-36%) (Figure 4D), indicating proximity between HSP70 and HbS but not HSP70 and HbA. Moreover, PLA+ SCD cells showed higher mean flu- orescence intensity under hypoxia than under normoxia (Figure 4D), indicating that hypoxia induces the formation of more potent HbS-HSP70 complexes that could account for cytoplasmic retention of HSP70.
In order to explore the potential of HbS polymers and HSP70 interacting within the same protein complex, we performed co-immunoprecipitation assays. SCD RBC sus- pensions were placed at normoxia or hypoxia then lysed, and HSP70 was immunoprecipitated. Using an anti-α-glo- bin antibody we found co-immunoprecipitation of HSP70 and α-globin under hypoxia but not under normoxia (Figure 4E) supporting the presence of HbS-HSP70 protein complex. Of note, despite using the same amounts of RBC for both conditions, there was less immunoprecipitated HSP70 under hypoxia than normoxia likely due to decreased solubility of HbS polymer fibers under hypoxia as evidenced by the color of the lysates under both condi- tions and the presence of a small red colored precipitate under hypoxia (Online Supplementary Figure S3).
Cell death during the terminal stages of erythroid differentiation in bone marrow of sickle cell disease patients
In order to validate the direct relevance of the in vitro findings to in vivo conditions, we analyzed terminal ery- throid differentiation using unmanipulated marrow sam- ples from SCD patients. Bone marrow aspirates were obtained from five SCD patients and cells were stained for surface markers GPA, CD49d and Band 3 and analyzed by
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Figure 1. (continued from previous page) (E) Percentage of enucleation meas- ured at D11 for CN, CH, PN and PH erythroblasts (means ± SEM; CN and CH: n=3; PN and PH: n=6). (F) Flow cytometry plots showing percentage of apoptotic cells (Annexin V-positive cells) measured at D3, D5, D7, D9 and D11. (G) Percentage of apoptotic cells in control (n=4) and patients (n=6) under normoxia (N) and hypoxia (H) at D7 and D9 of phase II of culture. *P<0.05, **P<0.01; Wilcoxon paired test and Mann-Whitney test (E and G).
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