Per2 inhibits cell proliferation. (A) K562 and U937 cells were cotransfected with either an empty expression vector (Con) or a V5-tagged Per2 expression vector (Per2-V5) along with a vector expressing the puromycin-resistant gene. Following 3 days of antibiotic selection, the surviving cells were harvested and analyzed by Western blotting with a V5 antibody. The blots were stripped and rehybridized with a GAPDH antibody. (B) Growth curve. K562 and U937 cells transfected with either empty vector (K562/Con and U937/Con) or Per2 expression vector (K562/Per2 and U937/Per2) were grown in fresh media. Aliquots were taken at 24-hour intervals for assessment of total viable cells. Data represent the mean ± SD of duplicate experiments. (C) K562 cells stably transfected with either a zinc-inducible V5-tagged Per2 expression vector (pMTPer2-V5) or an empty vector (pMT) were incubated with zinc for 16 hours and analyzed by Western blot with V5 antibody. The blot was stripped and rehybridized with a GAPDH antibody. (D) MTT assay. Equal numbers (3 × 104/mL) of K562 cells stably transfected with either empty vector (pMT) or a Per2 expression vector (pMTPer2) were grown in the presence of zinc. After 5 days, cell proliferation was determined by MTT assays. Untreated, wild-type K562 cells (wt) were included as control. Data are expressed as the mean ± SD of quadruplicate samples. The experiment was repeated twice.
