Tax-associated DDSB occur in S phase during DNA replication.

<p>A) HTLV-I-transformed MT-2 cells were pulse-labeled with BrDU to identify cells in S phase of the cell cycle. Immunofluorescence experiments indicated that γ-H2AX foci are detected in BrDU positive cells only. Cells were counter stained with DAPI. B) HTLV-I-transformed MT-2 cells were dual stained with Cyclin A, a marker of S phase, and γ-H2AX, a marker of DDSB foci. Cells were counter-stained with DAPI. C) HTLV-I-transformed MT-2 cells were dual stained with PCNA, which appear as a punctuated pattern in S phase, and γ-H2AX, to reveal DDSB foci. Cells were counter-stained with DAPI. D) Accumulation of DDSB foci in S phase was confirmed in Tax-only expressing cells using Tax-inducible JPX9 cells and Jurkat control, both treated with CdCl and dual-stained with Cyclin A and γ-H2AX antibodies. E) JPX9 cells were synchronized in G0/G1 by exposure to hydroxyurea (HU) overnight. G0/G1 JPX9 cells and non-synchronized JPX9 cells were exposed to CdCl to induce Tax expression and analyzed for presence of γ-H2AX DDSB foci. F) Efficacy of HU treatment was demonstrated by cell cycle analyses by FACS after PI staining. From top to bottom, untreated cells, HU G1-arrested cells, HU G1-arrested cells released by washing out HU and culturing 20 hours. G) Western blot analyses confirmed that HU treatment did not affect Tax expression.