Involvement of Caveolin-1 in Repair of DNA Damage through Both Homologous Recombination and Non-Homologous End Joining
Figure 9
Expression of Cav-1 expression contributes to the activity of the NHEJ repair pathways.
(A) MDA-MB-468 cells with or without silencing of Cav-1 were irradiated (5 Gy) for the indicated periods of time, and then subjected to Western blot analysis of phosphor-DNA-PKcs and total DNA-PKcs. (B) Left panel: HEK 293 cells containing a GFP-based chromosomal reporter, EJ5-GFP, were transfected with a caveolin-1 expression vector or a control empty vector. Thirty-six hours later, the cells were transfected with an HA tagged I-SceI endonuclease expression vector or an empty vector. Expressions of Cav-1 and HA-I-SceI were determined by Western blot. Right panel: Seventy-two hours following transfection with the HA-I-I-SceI plasmid, percentage of EGFP expressing cells, which represents the frequency of NHEJ, were determined by flow cytometry. The results shown are the mean ± S.E. from three identical experiments. (C) MDA-MB-468 cells with or without silencing of Cav-1were irradiated (5 Gy) for the indicated periods of time, and then fixed for immunostaining with Cav-1 and EGFR antibodies. Cav-1 staining was shown in green and EGFR staining in red. DAPI was used for nucleus staining. (D) MDA-MB-468 cells were irradiated (5 Gy) for the indicated periods of time. At the end of IR, cells lysates were prepared and subjected to immunoprecipitation and immunoblotting with either Cav-1 or EGFR antibodies as indicated. The light chain and heavy chains were used as loading controls.