Figure 3.

Exogenous SAMHD1 expression induces apoptosis and caspase-3/7 activity via activation of extrinsic apoptosis signaling. HuT78 vector control and SAMHD1 expressing cells were seeded at density of 2 × 10⁴ cells per 6-cm dish containing 5 ml growth media on day 0 following trypan-blue exclusion method. (A) Seven days post-seeding, cells in triplicate, were stained with propidium iodide (PI) and cell cycle analysis was performed via flow cytometry. Percentages of cells in G1/G0, S, and G2/M phases of cell cycle are presented. (B) Percentage of cells in Sub-G1 phase are presented. (C) HuT78 vector control and SAMHD1-expressing cells were seeded at density of 2 × 10⁴ cells per 6-cm dish containing 5 ml growth media on day 0 following trypan-blue exclusion method. Seven days post-seeding, cells were stained with annexin V-PE and 7-AAD in triplicate and analyzed by flow cytometry. Representative flow cytometric profiles are presented (left panel). The percentages of non-apoptotic cells, early apoptotic cells, and late apoptotic cells were quantified as presented (right panel). (D) On day 7 post-seeding, cell lysates from all cell lines were collected and western analysis was performed using the antibodies to caspase-8, caspase-9, caspase-3, PARP, HA (SAMHD1), and GAPDH (loading control). (E) On day 7 post-seeding, 1 × 10⁴ cells per cell line were collected in 4 replicates and incubated in 100 µl of Caspase-Glo 3/7 reagent at room temperature in dark for 1 hour. Caspase-3/7 activity was then determined by measuring luminescence values. All the data presented are representative of 3 independent experiments. B, C, E, ***, p < 0.001.