A TfR2 knockdown retards human erythroid progenitor differentiation. (A) Flow cytometric analysis of cell surface TfR2 expression in CD36⁺ cells at day 4 of the culture expressing a control shRNA (solid red), TfR2 shRNA A (blue line), TfR2 shRNA B (green line). The black line in the top panels corresponds to an isotypic control labeling. The top panels show a typical experiment, the bottom panel presents the results of 4 independent experiments showing the decrease of mean fluorescence intensity due to TfR2 knockdown. (B) Top panel, percentage of hemoglobinized cells determined by benzidine staining of day 5, 7, 9, and 12 cultures after CD36⁺ cell sorting. Bottom panel, expression levels of TfR1 (CD71) and GPA measured by flow cytometric analysis of control and TfR2 knockdown cells on culture day 4. R1, R2, R3, and R4 populations were determined as described previously.³⁴  (C) Evaluation of erythroid differentiation on days 5, 7, 9, and 12 of culture after CD36⁺ cell sorting. Cell classifications were established after May-Grunwald Giemsa staining of cytospin preparations.