SOX7 expression in K562 cell line inhibited cell proliferation. (A) Overexpression of SOX7 protein in K562 was confirmed by western blot analysis. (B) SOX7 expression reduced the rate of cell growth in culture. Cell number was enumerated on days 0, 3, and 6 on the basis of trypan blue exclusion (n = 3 separate experiments). (C) SOX7 expression significantly reduced 3[H] thymidine uptake (n = 3 separate experiments). The 3[H] thymidine-incorporation assay was performed in which 0.1 × 10⁶ transduced K562 cells were incubated in 200 μL of 3[H]thymidine-containing culture medium at 0.025 mCi/mL for 18 hours. Cells were applied on a Cytostar-T scintillating microplate (Perkin Elmer, Waltham, MA) using vacuum suction, dried at 55°C in an incubator overnight, and scintillant was thereafter added to the microplate. The counts-per-minute (cpm) reading was recorded using TopCount NXT (Perkin Elmer). (D) SOX7 expression significantly reduced the rate of cell division as shown by SNARF-1 staining (n = 4 separate experiments). Flow cytometric histograms on day 0 (white area) and day 2 (green area) were superimposed (inset). (E) GFP-SOX7 expression (upper) delayed cell cycle progression in S and G₂/M phases (n = 3 separate experiments). Comparisons between GFP and GFP-SOX7: G1, P = .004; S/G2/M, P = .011. SOX7 expression (lower) had no effect on apoptosis on the basis of 7-aminoactinomycin D (7AAD)/annexin V staining. Representative records of 2 separate experiments. (F-G) SOX7 expression reduced the clonogenic activity of K562 cells. GFP and GFP-SOX7 expression was confirmed by the presence of green fluorescence (F), and SOX7 expression significantly reduced colony frequency (G) (n = 4 separate experiments). Clonogenic assay was performed by plating transduced K562 cells onto 35-mm tissue culture plates in triplicates of 1.0 × 10³ cells/mL in MethoCult GF H4230 supplemented with 10% fetal bovine serum (STEMCELL Technologies, Vancouver, BC, Canada). Colonies were enumerated on day 12 after culture. Scale bars represent 1.0 mm.