Fig. 3

NSC 19630 induces apoptosis in HTLV-1-transformed and patient-derived cells. a ED and MT-4 cells were exposed to WRN helicase inhibitor NSC 19630 (3 μM) or DMSO. After 72 h, cells were stained with annexin V. The figures include the percentage of cells in the four quarters: Q1, Q2, Q3, and Q4. Q3 included the live cells that are annexin V and PI negative. Q4 included early apoptotic cells, which are annexin V positive and PI negative. Q2 included cells in late apoptosis, which are both annexin V and PI positive. Finally, Q1 included necrotic cells, which are PI positive and annexin V negative. b Western blots of Tax viral protein in ED, C8166, C91PL, and MT-4 cells exposed to NSC 19630 compared to DMSO-treated controls. c HTLV-1-transformed cell lines (MT-4, C8166, C91PL, and 1186.94) and patient-derived cell lines (ED, TL, ATL-25, ATL-43T, ATL-55T, LMY1, KK1, SO4, and KOB) were treated for 72 h with 3 μM of NSC 19630. Cells were stained with annexin V to measure the apoptotic effect of the WRN helicase inhibitor. The percentage of apoptosis and necrosis was graphed. HTLV-1-transformed, ATL-derived IL-2-dependent cell lines and IL-2-independent cell lines are represented in blue, yellow, and red, respectively. Tax viral protein is expressed in MT-4, C8166, C91PL, 1186.94, and ATL-25 cell lines [23]. Experiments were performed multiple times in duplicate. Representative results are shown in the final figures. d Western blot of Caspase-3 and apoptotic markers Bcl-2 and Mcl-1 was performed in ED, ATL-55T, and LMY1 cells exposed to DMSO or 3 μM of NSC 19630. Our analysis shows the activation of caspase-3 after treatment with the WRN helicase inhibitor. e Distraction of mitochondrial transmembrane potential in ED cells treated with NSC 19630 compared to DMSO control. f Immunofluorescence of ɣ-H2AX and PCNA in ED cells exposed for 72 h with 3 μM of NSC 19630 compared to DMSO control. Fluorescent images were captured by using the ×100 objective