Fig. 3

MGMT interacted with BRCA1 in NPC cells with CDDP treatment. After treatment with or without 10 µM CDDP for 8 h, the protein lysates of a HONE-1 and b TW01 cells were subjected to Co-IP analyses with 1 µg/mL anti-MGMT antibodies followed by Western blotting. Following the indicated treatment as in a and b, representative immunofluorescence images of DAPI, MGMT, and BRCA1 in c HONE-1 and d TW01 cells were processed through confocal microscopy. At least 200 cells were subjected to each treatment in each experiment. The bar graph (right panel) shows the percentage of tested cells containing five or more colocalizing foci for the staining proteins. Bar values are presented as mean ± SD of at least three independent experiments. *P < 0.05. e Endogenous physical interactions between MGMT and BRCA1proteins were detected by using in situ PLA in HONE-1 cells with or without CDDP treatment (10 µM for 8 h). PLA-positive signals are indicated by red fluorescent puncta and visualized by fluorescence microscopy. DAPI was used to detect the nuclei. To examine nuclear proteins, HONE-1 cells were initially f treated with O6BG (60 μM) or g transfected with MGMT-targeted siRNA. Following CDDP treatment as indicated in a, nuclear protein lysates were extracted and subjected to Western blot analyses with anti-pRBCA1, BRCA1 and MGMT antibodies. Fold changes in protein levels listed under each blot were normalized to the levels of the actin control. Representative results of at least three independent experiments are shown. Scale bars indicate 10 µm