Figure 4

dRif1 does not colocalize to the DNA damage foci induced by HU and Aphidicolin. A) S2 cells were treated with either 2.5 mM hydroxy Urea (HU) or 25 μM aphidicolin for 16 hrs to induce DNA damage and then fixed, stained with antibodies to dRif1 (red) and γH2AvD (green). The slides were mounted in mounting media containing DAPI or TO-PRO 3 (blue). B) S2 cells were treated with either dsRNA of GFP (1) or dRif1 (2). Each was further split into three parts and was either mock treated or treated with hydroxyurea (HU) or bleomycin. Protein extracts were tested for dRif1 and γH2AvD expression; tubulin was used as a loading control. dRif1 was undetectable in RNAi treated cells. γH2AvD levels increase upon exposure to DNA damage. C) The cells were also immunostained with γH2AvD to detect DNA damage sites. All the cells were fixed and stained for dRif1 (red), γH2AvD (green) antibody and DAPI (blue) Scale bar is 5 μm.