A caspase-mediated cleavage of GATA-1 in MDS erythroblasts. (A) GATA-1 gene expression. RT-qPCR for GATA1 transcripts in MDS versus control erythroblasts. Normalized relative quantity (n_RQ) to HPRT. Median values indicated as horizontal bars. (B) Apoptosis. Flow cytometry for annexin V⁺/7-AAD⁻ cells in MDS (n = 30; ■) and control (n = 22; ○) erythroblasts. Mean percentages ± SE. (C) Caspase-3 activity. Flow cytometry for caspase-3 like activity in MDS (n = 8; ■), pan-caspase inhibitor Q-VD-OPH (100μM)–treated MDS (n = 1; ) and control (n = 4; ○) erythroblasts. Results as mean fluorescence intensity. (D) GATA-1 protein expression. Nuclear fraction proteins blotted with the indicated Abs (top panel). Quantification of GATA-1 by densitometry expressed as a ratio to Hsc70, normalized to the ratio of GATA-1 to Hsc70 in UT-7 control cell line for each blot (bottom panel) in MDS with (n = 11) or without (n = 1) dyserythropoiesis and 8 controls. (E) Inhibition of caspases rescues GATA-1 expression. Q-VD-OPH (100μM) or MG132 (20μM) is added on MDS and control cultures. Immunoblot for GATA-1 in whole-cell lysates. Actin or Hsc70 are used as loading controls. Student t test for P value; *P < .05; **P < .01; ns indicates not significant.