Ectopic expression of THAP1 into primary human ECs inhibits cell-cycle progression and blocks S-phase DNA synthesis. (A) Inhibition of DNA synthesis in ECs expressing THAP1. HUVECs transduced with pMLV-MCS (HUVEC-MCS) or pMLV-THAP1 (HUVEC-THAP1) retroviral expression vectors were pulse-labeled with BrdU for 90 minutes. Cy2-conjugated anti-BrdU antibody was used to identify cells incorporating BrdU in the S phase of the cell cycle. Cells were visualized for BrdU incorporation (top panels) and THAP1 expression (bottom panels). (B) The graph shows the percentage of BrdU⁺ cells in HUVEC, HUVEC-MCS, and HUVEC-THAP1 cell populations. At least 500 cells were counted for each condition. Results are the mean of 2 independent retroviral transduction experiments. (C) Down-regulation of nuclear proliferation marker Ki-67 in ECs expressing THAP1. The graph shows the percentage of Ki-67⁺ cells in HUVEC-MCS and HUVEC-THAP1 cell populations. Ki-67 is a marker of cycling cells in late G1 and S-G2/M cell-cycle phases; therefore, reduction in the number of Ki-67⁺ cells in HUVEC-THAP1 indicates an inhibition of cell-cycle progression in ECs expressing THAP1. (D) Flow cytometry analysis of cell-cycle distribution in HUVEC-MCS and HUVEC-THAP1 cell populations. Black area represents cells in S phase, and red area represents cells in G1 and G2/M phases. (E) Ectopic expression of THAP1 into HUVECs inhibits cell-cycle progression at the G1/S transition. The graph shows the percentage of cells in G1, S, and G2/M phases of the cell cycle in HUVEC-MCS and HUVEC-THAP1 cell populations. Results are the mean of 2 independent retroviral transduction experiments. (F) Ectopic expression of THAP1 into human U2OS osteosarcoma cancer cells inhibits S-phase DNA synthesis. The graph shows the percentage of BrdU⁺ cells in control untransfected cells or in U2OS cell populations expressing THAP1 (% BrdU⁺ cells in the THAP1⁺ population) or the unrelated nuclear factor PAPSS1 (% BrdU⁺ cells in the PAPSS1⁺ population). Analysis was performed 48 hours after transfection. (G) Ectopic expression of THAP1 impairs growth of U2OS cells. Cells transfected with indicated pcDNA3 expression vectors were selected in neomycin for 14 days prior to crystal violet staining. Error bars in panels B, C, E, and F indicate SD.