Fig. 4

Overexpression of USP37 leads to enhanced survival in response to replication stress while its depletion leads to reduced survival in osteosarcoma cells with increased accumulation of USP37 in cytoplasm. A The clonogenic potential was examined by Colony formation assay. U2OS osteosarcoma cells were transfected with Myc USP37 for overexpressing USP37 while SiRNA was used to reduce expression of USP37, following which cells were treated with different concentrationsn of HU (Hydroxy Urea) for 24 h., then washed and fresh media was added. Subsequently colonies were allowed to develop for 7 days. B Osteosarcoma cell U20S cells were treated with 300 µM HU for 24 h and processed for IF to see USP37 expression. C Osteosarcoma cell line MG-63 was treated with 300 µM HU for 24 h. and processed for IF to see USP37 expression. D Western blotting to see the expression of USP37 and corresponding DNA damage marker ϒH2AX after induction of replication stress in U2OS whole cell lysate. E Nuclear and cytoplasmic fractionation was carried out on U2OS cells after treatment by an escalating dose of HU and western blotting was done to assess the level of USP37 in the nuclear and cytoplasmic fraction