SOCS3 activation mediates inhibition of cell proliferation. (A-C) SOCS3 overexpression in CD34⁺-derived erythroblasts. (A) Growth curve of cultures infected with the empty vector (EV; ♦), the Sox6-overexpressing vector (■), or the SOCS3 overexpression vector (▴). The y-axis indicates the number of cells; x-axis, days in culture (all cultures were transduced at day 6 with similar efficiency). (B) Differential cell counts. The different colors in the columns represent the proportion of mature (orthochromatic plus polychromatophils; O+P) and immature (basophils plus proerythroblasts) cells, respectively. EV-transduced cells: O+P = 17%; Sox6-transduced cells: O+P = 43%; SOCS3-transduced cells: O+P = 24%. (C) Real-time PCR on Bcl-2 and Bcl-xL expression, 48 hours after transduction of day 6 erythroblasts with EV, Sox6, and SOCS3, respectively. Histograms show the relative levels of expression (mean ± SEM of at least 3 independent experiments) compared with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) considered as 1. The differences shown were statistically significant, as indicated by P values. (D-F) Transduction experiments in HEL cells. (D) Neither Sox6 nor SOCS3 overexpression altered cell proliferation. Growth curve (left) and percentage of GFP⁺ cells (right) of HEL cells transduced with the empty control vector (EV-HEL), the Sox6-overexpressing vector (Sox6-HEL), and the SOCS3-overexpressing vector (SOCS3-HEL). (E) Quantitation by real-time PCR of SOCS3 expression in HEL cells transduced as above, relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (F) Real-time PCR. Only Sox6 activated erythroid gene transcription.