Thymidine

When applicable, a double thymidine block was used to synchronize the cell cycle and to synchronize the initiation of reactivation and increase efficacy of viral reactivation (70 (link)). TREx K-Rta BCBL-1 and BCBL-1 cells at 30% confluence were incubated with 2 mM thymidine (Sigma-Aldrich) for 18 h. The cells were washed with fresh medium to remove the thymidine and allowed to grow for 9 h. The cells were again treated with 2 mM thymidine for 18 h. After the second thymidine treatment, the cells were washed with medium to remove thymidine, and KSHV reactivation was induced for 4 h with various stimuli described in the figure legend.

S phase cell cycle synchronisation was achieved by double-thymidine treatment, consisting of 2 mM thymidine (#T1895, Merck, Kenilworth, NJ, USA) for 14.5 h treatment, released for 9 h, followed by 2 mM thymidine treatment for additional 16 h. No release time was included between double-thymidine treatment and nucleofection. Cell cycle synchronisation at the G2/M phase was achieved using nocodazole (#M1404, Merck, Kenilworth, NJ, USA) at 11 µM final concentration for 16–16.5 h at 37 °C. Cultures were released from nocodazole treatment for 40–120 min prior to nucleofection. Non-synchronised cultures were maintained in parallel under standard conditions.

For double-thymidine blocks, 1.5 × 10⁵ cells/well were plated in 6-well plates the day before siRNA transfection. After 6 h, the cells were washed and placed overnight in a medium supplemented with 2 mM of thymidine (Sigma) for 14–17 h. After extensive washes, cells were placed in a fresh medium for 10–12 h before the second thymidine block for 14–17 h. Cells were released and harvested at 0, 3, 6, 9, or 12 h for cell cycle analysis. On average, 90% of cells were in G₁/S at the time of release. To follow cells progressing through S phase, cells were pulse-labeled with EdU for 60 min before harvesting. For thymidine-nocodazole blocks, U2OS cells in log phase were treated for 14–15 h with 2 mM of thymidine and released for 6 h before 250 ng/ml of nocodazole (Sigma) was added to the medium for another 6 h. Cells were released and harvested at 0, 1, 3, 7, or 12 h after extensive washes. For caffeine and UCN-01 treatments, LSD1 siRNA-treated cells were incubated with 5 mM of caffeine (Sigma) dissolved in water at 80 °C at a concentration of 100 mM for 2 h or with 500 nM of UCN-01 (Sigma) dissolved in DMSO at 100 mM for 2 h before a 60-min pulse with 10 μM of EdU for cell cycle analysis by EdU/PI incorporation.

To obtain cell populations synchronised at G1/S or in early S, late S, or G2, A549 were subject to double-thymidine block (2 mM thymidine (Sigma) for 18 h, released into fresh media for 8 h, arrested with thymidine for 17 h) then lysed directly or released into full medium for 2.5, 5.5, or 7.5 h. To obtain cell populations synchronised at G2/M or in M, or early G1, A549 were subject to a thymidine/nocodazole block (2 mM thymidine for 24 h, released into full medium containing 100ng/ml nocodazole (Sigma) for 14 h) then mitotic cells were collected by knocking off the dish, and either lysed directly or replated into full medium for 0.5 or 4 h. Propidium iodide staining with flow cytometry analysis, and immunoblotting for a panel of stage-specific cell cycle markers, are routinely used to confirm enrichment of cell cycle phases. For analysis of each timepoint, adherent cells were trypsinised and pooled with non-adherent cells from the medium, and then processed for RNA or protein extraction.

To synchronize in G1/S phase, double thymidine block was accomplished by growing cells in the medium containing 2 mM thymidine (Sigma-Aldrich) for 18 h, released into fresh media without thymidine for 9 h. The cells were then reincubated in the medium containing 2 mM thymidine for an additional 17 h. Next, cells were released into fresh media for 2 h for synchronization in S-phase. The synchronized cells in S-phase were lysed in RIPA buffer, and total proteins were analyzed by western blotting using H3 and H4K12ac antibodies.