HSP70 overexpression rescued GATA1 expression, decreased ROS production, limited free heme content, and rescued the heme/globin balance. (A) Relative protein expression of GATA1, HSP70, β-globin chain, TfR1, and RPL11 after depletion of RPS19 or RPL11 in primary erythroid cells and rescue with overexpression of wild-type HSP70 cDNA. Data at day 9 of terminal erythroid culture, obtained from immunoblots of 100 000 human erythroid primary cells derived from CD34⁺ cells from cord blood and depleted in RPS19 or RPL11 by specific shRNAs. Proteins are compared with β-actin. The data are relative to the protein expression levels in the shcontrol for each protein studied (value = 1). (B) Relative mRNA expression compared with β-actin and shcontrol gene expression (value = 1) after depletion of erythroid cells in RPS19 (left panel) or RPL11 (right panel) mRNA after cord blood CD34⁺ cell lentiviral infection by specific shRNAs and rescue with overexpression of wild-type HSP70 cDNA. The data are mean ± standard deviation of 3 independent experiments and correspond to the relative mRNA expression of each gene (HSP70, GATA1, α and β globin, ALAS2, FECH, TfR1, EIF2α, HRI, RPS19, and RPL11) compared with the reporter genes, such β-actin, and the shcontrol (value = 1). No significant effect of HSP70 overexpression has been noted in depleted RPS19 erythroid cells (left panel), whereas a significant increase in all mRNAs tested, with the exception of RPS19 and RPL11 mRNA, was found after HSP70 overexpression in depleted RPL11 erythroid cells (right panel). (C) HSP70 overexpression rescued the FLVCR1 increase in depleted RPS19 erythroid cells and, to a greater extent, in depleted RPL11 erythroid cells. (D) HSP70 overexpression significantly increased erythroid cell survival in depleted RPL11 erythroid cells. Cell mortality was assessed with 0.2% trypan blue. The data are mean ± standard deviation of 3 independent experiments. *P < .05, ***P ≤ .001.