Figure 7.

The PWWP domain is indispensable for EXPAND1 functions.

a) Generation of siRNA-resistant EXPAND1 constructs. 293T cells were co-transfected with indicated constructs and siRNAs. 24 post transfection, cells were lysed and immunoblotting experiment was done using indicated antibodies, b-d) PWWP is required for EXPAND1-dependent chromatin condensation. Stable cells expressing siRNA#1-resistant EXPAND1 full-length (FL), its D1 or dPWWP mutants were treated with siRNA#1 twice at 24 hr interval. Cells were incubated with 200 ng/ml NCS for 1 hr before nuclei were extracted. Microccocal nuclease sensitivity was assessed as described in Methods and Materials. Expression of siRNA-resistant EXPAND1-expressing cells are shown (b), Microccocal nuclease sensitivity assay was displayed in (c) and average size of nucleosomes is shown in (d), e) Stable cells as described in (b) were treated with EXPAND siRNA#1 twice at 24 hr intervals. 24 hr after the second transfection, cells were seeded onto coverslips and irradiated (3 Gy) 24 hr later. At indicated time points cells were fixed and processed for immunostaining using pH2AX antibodies, f) clonogenic assay of EXPAND1-expressing cells following radiation. EXPAND1 siRNA-transfected cells were seeded onto 60 mm dish and were irradiated with indicated dose. Cells were incubated for 14 days before being fixed and counted. Results represent mean±S.D. from two independent experiments each done in triplicate.