Silencing of Cav-1 expression reduces the DSB repair by HR.

<p>(<b>A</b>) HT-1080 cells were irradiated (5 Gy) for the indicated period of time, and then cell lysates were prepared for Western blot analysis of Cav-1 and γ-H₂AX. β-actin was used as a loading control. (<b>B</b>) To determine the turnover of the silencing effect of Cav-1 siRNA in HT-1080 cells, we performed Western blot of analysis of Cav-1 at the indicated time after siRNA transfection. (<b>C</b>) HT-1080 cells were transfected with a non-targeting RNA or Cav-1 siRNA. Twenty-four hours after transfection, the cells were transfected with an HA tagged I-SceI endonuclease expressing vector (HA-I-SceI) or empty vector (HA) by electroporation, followed by Western blot analysis of Cav-1 and HA-I-SceI (upper panels), and by puromycin screening for HR frequency (lower panels). HR frequency was calculated as follows: the average numbers of colonies/dish were divided by the plating efficiency of transfection and divided by 85,000 (the total number of cells plated). The results shown are the representative of three similar experiments; each bar represents the mean±S.D. of triplicate determinations.