Figure 4.
HSP70 overexpression rescued erythroid proliferation and differentiation and decreased the apoptosis of erythroid cells in mutated RPL11+/MutDBA patients. (A) Study of erythroid proliferation (number of cells ×104) between day 6 and days 14 or 15 from CD34+ peripheral blood from various DBA-affected patients carrying a mutation in RPS19 gene (UPN#37) (middle and bottom left plots) or RPL11 gene (UPN#172 (top and middle right plots), UPN#189 (red dot), and UPN#1130 (yellow dot) (bottom right plot) compared with healthy controls (Co) (open circle) or CD34+ cells infected with WT HSP70 cDNA lentivirus (filled circle) (top left plot). Erythroid cells from DBA affected patients have been analyzed with or without WT HSP70 cDNA lentivirus infection. Counting of the erythroid cells has been performed using flow cytometry (shown) and manual count under the microscope in triplicate. Five thousand CD34+ cells have been plated at day 0. Erythroid proliferation has been calculated compared with this number of cells during the time course at days 9, 12, and 14 or 15. (B) Top panel: western blot is shown from the erythroid cells (20 000 cells) obtained at day 10 from CD34+ peripheral blood from a DBA affected patient carrying a mutation in RPL11 gene (UPN#479). CD34+ DBA patients’ cells in erythroid differentiation have been studied without or with wild-type HSP70 cDNA lentivirus infection. HSP70, GATA1, procaspase-3 and β-globin protein expressions have been studied compared with β-actin expression level. Bottom panel: a RPL11+/Mut DBA affected patient exhibited a defect in α-globin and β-globin expression level in accordance with the hemoglobin concentration of the patient. Overexpression of wild-type cDNA HSP70 in erythroid cells from the RPL11+/Mut UPN#172 DBA affected patient restored α-globin and β-globin expression in this patient compared with the normal erythroid cells transduced with wild-type cDNA HSP70. Globin gene expression level has been measured by imaging flow cytometry (ImageStream). (C-D) Representative FACS histogram plots of cultured erythroid cells (10 000 cells) from a healthy control (Co) (left panels), a RPL11+/Mut DBA patient (UPN#1099) (middle panels), and this patient after wild-type HSP70 cDNA lentivirus infection. (C) Apoptosis analysis on the DAPI and annexin V labeling. DAPI−/annexin V+ apoptotic erythroid cells have been quantified in percentage. (D) Erythroid differentiation analysis on the CD34/CD36 labeling. In all the FACS histogram plots, the ordinate and the abscissa measured the number of cells displaying the fluorescent intensity of the different antibody staining.

HSP70 overexpression rescued erythroid proliferation and differentiation and decreased the apoptosis of erythroid cells in mutated RPL11+/MutDBA patients. (A) Study of erythroid proliferation (number of cells ×104) between day 6 and days 14 or 15 from CD34+ peripheral blood from various DBA-affected patients carrying a mutation in RPS19 gene (UPN#37) (middle and bottom left plots) or RPL11 gene (UPN#172 (top and middle right plots), UPN#189 (red dot), and UPN#1130 (yellow dot) (bottom right plot) compared with healthy controls (Co) (open circle) or CD34+ cells infected with WT HSP70 cDNA lentivirus (filled circle) (top left plot). Erythroid cells from DBA affected patients have been analyzed with or without WT HSP70 cDNA lentivirus infection. Counting of the erythroid cells has been performed using flow cytometry (shown) and manual count under the microscope in triplicate. Five thousand CD34+ cells have been plated at day 0. Erythroid proliferation has been calculated compared with this number of cells during the time course at days 9, 12, and 14 or 15. (B) Top panel: western blot is shown from the erythroid cells (20 000 cells) obtained at day 10 from CD34+ peripheral blood from a DBA affected patient carrying a mutation in RPL11 gene (UPN#479). CD34+ DBA patients’ cells in erythroid differentiation have been studied without or with wild-type HSP70 cDNA lentivirus infection. HSP70, GATA1, procaspase-3 and β-globin protein expressions have been studied compared with β-actin expression level. Bottom panel: a RPL11+/Mut DBA affected patient exhibited a defect in α-globin and β-globin expression level in accordance with the hemoglobin concentration of the patient. Overexpression of wild-type cDNA HSP70 in erythroid cells from the RPL11+/Mut UPN#172 DBA affected patient restored α-globin and β-globin expression in this patient compared with the normal erythroid cells transduced with wild-type cDNA HSP70. Globin gene expression level has been measured by imaging flow cytometry (ImageStream). (C-D) Representative FACS histogram plots of cultured erythroid cells (10 000 cells) from a healthy control (Co) (left panels), a RPL11+/Mut DBA patient (UPN#1099) (middle panels), and this patient after wild-type HSP70 cDNA lentivirus infection. (C) Apoptosis analysis on the DAPI and annexin V labeling. DAPI−/annexin V+ apoptotic erythroid cells have been quantified in percentage. (D) Erythroid differentiation analysis on the CD34/CD36 labeling. In all the FACS histogram plots, the ordinate and the abscissa measured the number of cells displaying the fluorescent intensity of the different antibody staining.

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