Journal: Genes

Article Title: Role of RPA Phosphorylation in the ATR-Dependent G2 Cell Cycle Checkpoint

doi: 10.3390/genes14122205

Figure Lengend Snippet: ( A ) WT and S4A/S8A-expressing cells treated with etoposide (20 µM) for 2 h (-2 indicates start of etoposide treatment) followed by drug removal (0 h) for the indicated times were assessed for chromatin retention in S phase (upper panel) and G2 phase cells (lower panel). Orc2 was used as a loading control in each case. ( B ) Immunofluorescence of cells that were detergent washed before fixation. Foci formation images comparing the chromatin accumulation of TopBP1 and RPA32 for etoposide (20 µM) and mock-treated cells at 4 h after etoposide removal. ( C ) Calculated percentage of cells containing RPA and TopBP1 foci in a total of 200 cells per condition for each experiment.

Article Snippet: Cell synchronized in G2, non-treated and treated with etoposide as described and fixed at 4 h. After an initial wash with PBS, cells were extracted with PBS containing 0.5% Triton X-100 for 2 min on ice and fixed with 4% paraformaldehyde for 15 min. Next, the coverslips were blocked with 15% goat serum at room temperature and then incubated with primary antibodies to HA (Covance, Princeton, NJ, USA), TopBP1, PS33-RPA32 (Bethyl Laboratories) or CENP-F (Santa Cruz) in blocking solution for 1 h. The coverslips were washed with PBS and incubated with an appropriate Alexa-Fluor 488- or Alexa-Fluor 568-conjugated antibody in blocking solution for 1 h. Cells were mounted in PermaFluor (Fisher) supplemented with 0.5 μg/mL DAPI (Roche, Basel, Switzerland).

Techniques: Expressing, Control, Immunofluorescence