Sox6 overexpression in K562 cells. (A) Schematic representation of the lentiviral vector used. LTR indicates Long Terminal Repeats; SFFV, Spleen Focus Forming Virus; WPRE, Woodchuck-Hepatitus-Virus-Posttranscriptoral-Regulatory Element. (B) Expression of the transduced Sox6 was assayed at the mRNA and protein level. (Top panel) Reverse-transcription PCR with primers detecting the exogenous, vector-derived Sox6 transcript. β-Actin primers were used as control. (Bottom panel) Western blot with the anti-FLAG antibody detected the exogenous Sox6 protein. The anti–β-actin antibody was used to normalize for protein loading. (C-E) Phenotypic changes of K562 cells on Sox6 overexpression. Sox6-K562 cells (right panels), compared with EV-K562 cells (left panels), showed (C) a reddish pellet that indicated the accumulation of hemoglobin chains, quantitated by enzyme-linked immunosorbent assay in the right panel; (D) an increased number of o-dianisidine–positive cells (brown staining), which indicated hemoglobin accumulation; and (E) increased GpA (CD235) positivity (FACS analysis; compare cells in R6 gate: 28.14%, multiplicity of infection [MFI] of 517.28 in Sox6-K562 vs 17.87%, multiplicity of infection of 384.47 in EV-K562). (F-H) Sox6-K562 cells stopped growing and underwent apoptosis: 1 × 10⁶ exponentially growing K562 cells were transduced at day 0 either with the Sox6 or the EV vector and were washed and replated in fresh medium 24 hours after transduction. (F) Sox6-transduced cells stopped growing 3 days after transduction, and the culture died by day 9. Error bars refer to 3 independent experiments. (G) Real-time PCR on Bcl-2 and Bcl-xL expression, 72 hours after transduction. 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. Statistical significance is indicated above the chart. (H) Seventy-two hours after transduction, FACS analysis was performed with anti–annexin V antibody and propidium iodide staining to evaluate apoptosis. In Sox6-K562 cells, 16.8% of cells were positive for both annexin V and propidium iodide, whereas only 7% of EV-K562 cells were double positive for the same markers.