Overexpression of wild-type (WT) HSP70 restored HSP70, GATA1, and erythroid proliferation in depleted RPL11 erythroid progenitors. (A) Erythroid proliferation (number of cells ×10⁴) during the time course from day 4 to day 11 of erythroid culture from normal cord blood CD34⁺ depleted in RPS19 or RPL11 by specific shRNAs has been studied compared with noninfected CD34⁺ cells (Co) or infected with various random scrambles (SCR-1 and SCR-2) or WT HSP70 cDNA lentivirus. The depleted RPS19 or RPL11 erythroid cell proliferation has been studied with and without WT HSP70 cDNA lentivirus infection. Five thousand CD34⁺ cells have been plated at day 4. Erythroid proliferation has been calculated compared with this number during the time course. Study of erythroid proliferation (number of cells ×10⁴) between day 5 and day 10 from synchronized BFU-E (B) and CFU-E (C) erythroid progenitors in RPS19 or RPL11 deficient erythroid cells compared with SCR infected cells or WT HSP70 cDNA lentivirus infection (left panels). The depleted RPS19 (middle panels) or RPL11 (right panels) erythroid cell proliferation has been studied with (black spots) and without (white spots) WT HSP70 cDNA lentivirus infection. Five thousand CD34⁺ cells have been plated at day 5. Erythroid proliferation has been calculated compared with this number during the time course at days 7 and 10. Counting of the erythroid cells has been performed using flow cytometry (shown) and manual count under the microscope in triplicate. The data are shown as the mean ± the standard deviation (n = 3 experiments). **P < .01; ***P < .001. Expression level of total HSP70 and its cytoplasmic and nuclear subcellular distribution (D) and nuclear expression of GATA1 (E) in deficient RPS19 (shRPS19) and RPL11 (shRPL11) erythroid cells after (dark blue and red curves, respectively) or not (light blue and orange curves, respectively) WT HSP70 cDNA lentivirus infection at day 10 of synchronized erythroid culture. The results are presented in MFI obtained by using imaging flow cytometry (ImageStream). The vertical green and red lines represented the MFI of HSP70 expression (D) and GATA1 (E) in erythroid cells infected with shSCR or WT HSP70 cDNA lentivirus in depleted RPL11 cells, respectively. The total efficiency of the HSP70 rescue is shown by the overlapping of both lines as shown on the total expression of HSP70 but also in nucleic GATA1 expression. (F) Imaging flow cytometry (ImageStream) analysis of GATA1 and HSP70 immunostainings in synchronized erythroid cells at day 7 of culture after depletion of RPS19-shRNA or RPL11-shRNA and overexpression or not of WT HSP70 cDNA. Shown are HSP70, GATA1, DAPI, and merged HSP70 and GATA1 stainings. Original magnification ×60. Scale bar represents 10 µm. (G) β-globin expression level in deficient RPL11 (shRPL11) erythroid cells after (red curve) or not (orange curve) WT HSP70 cDNA lentivirus rescue at day 10 of synchronized erythroid culture. The results are presented as the MFI obtained by using imaging flow cytometry (ImageStream).