Pd168393

30–40 larvae were dechorionated and incubated in 6-well plates at 29°C in either 10 µM solution of BrdU in 2% DMSO in E3 or only 2% DMSO in E3 for 2 hours. They were given three 5 minutes washes with E3, followed by PFA fixation and methanol up-gradation. After downgrading to PBS and prior to processing for immunostaining, the fixed larvae were treated with 4N HCl for 20 minutes at room temperature and processed for immunostainings.
For Hydroxyurea treatment, working concentration of 50 mM was prepared using E3 without methylene blue. In case of combined treatment, 30 mM hydroxyurea and 150 uM aphidicolin was used. Final concentration of DMSO was adjusted to −0.05% v/v to increase the permeability of Aphidicolin. Both Hydroxyurea and combined treatment of Hydroxyurea+Aphidicolin were done at 24hpf on dechorionated embryos. The 1 mM stock of PD168393 (Merck Millipore 513033) was prepared in DMSO and used at 10 µM final concentration in 1% DMSO in E3 starting from 18hpf.
The TUNEL staining was performed using Promega Kit as per the manufacturer's instructions. Briefly, the embryos at 48hpf were fixed in 4%PFA, washed in PBT (0.1 m phosphate buffer and 0.8% triton X-100), incubated with TUNEL reaction mixture at 37°C followed by washes, DAPI staining and mounted in Glycerol for imaging.

T84 cells were seeded in a 35-mm plastic dish on day 0. On day 1, 10 μM of Y-27632 (Sigma-Aldrich Co. LLC), 25 μM of blebbistatin (Enzo Life Sciences, Inc., Farmingdale, NY, USA), 1 μM of PD168393 (EMD Millipore, Billerica, MA, USA) or dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd, Osaka, Japan) was added. On day 4, the cell lysate was extracted for western blotting.