PU.1 induces growth arrest and apoptosis in L428 and KM-H2 cHL cell lines in vitro. (A) Western blot of PU.1 protein after tetracycline withdrawal. PU.1 was highly induced after tetracycline withdrawal in L428^tetPU.1 and KM-H2^tetPU.1 cells. PU.1 induced growth arrest in L428^tetPU.1 (B) and KM-H2^tetPU.1 cells (C) after tetracycline removal (○), whereas uninduced cells (●) grew comparably to wild-type parental cells. (D) Cell-cycle analysis was performed in L428^tetPU.1 and KM-H2^tetPU.1 cells by BrdU and 7-aminoactinomycin D staining. PU.1 induced G₁ arrest and led to a significant decrease in S-phase cells in L428^tetPU.1 and KM-H2^tetPU.1 cell lines. (E) PU.1 induced apoptosis in L428^tetPU.1 and KM-H2^tetPU.1 cells as assessed by annexin V staining. (F) PU.1 induced morphologic changes in L428^tetPU.1 and KM-H2^tetPU.1 cells. L428^tetPU.1 cells expressing PU.1 exhibited numerous different-sized cell processes and vacuoles compared with uninduced cells. A number of cells displayed nuclear fragmentation (Tet⁻, day 3, bottom left panel). The majority of KM-H2^tetPU.1 cells expressing PU.1 also contained relatively small-sized vacuoles and nuclear condensation, indicative of apoptosis. Images were acquired using a BX60 microscope and DP70 digital camera with DP controller software (Olympus; ×400 magnification).