Bag1 is required for I/11 erythroid differentiation. (A) Protein levels of Bag1 in I/11 erythroblasts 3 days after transduction with Sc shRNA lentivirus and 2 distinct shRNA lentiviruses complementary to Bag1. (B) Cumulative cell numbers of I/11 erythroblast cultures after transduction with control shRNA (▴) and shRNA against Bag1 (A and B ) Cumulative cell numbers are calculated from 3 independent experiments. (C) Cell size profiles, measured by a cell counter, from differentiating Bag1-deficient I/11 erythroblasts shown on the day of Epo induction (0h) and after 96 hours. Peaks at 10 μm indicate erythroblasts; peaks at 5 μm represent enucleated reticulocytes. (D) I/11 erythroblasts were transduced with lentiviral shRNA constructs as indicated. Cells were cultured for 3 days in proliferation conditions (top panels), followed by 4 days in differentiation conditions (bottom panels) before being harvested for cytospins and stained for hemoglobin (brown color) and histological dyes. Bars represent 25 μm.