FIG.7.

Estrogen protection of cells from APPct complex-induced apoptosis and its dependency on the ER. (A) N2a cells were transfected with 0.3 μg of pEGFP, Gal4-APPct, and HA-Fe65 together, with or without Myc-Tip60. After treatment with EOH or 10⁻⁸ M E2 for 72 h, the number of transfected (green) cells in each well was counted. Triplicate samples were analyzed for each data point, and the data were reproduced. (B) N2a cells transfected and treated as described for panel A were fixed and stained with DAPI. Representative micrographs are shown. (C) N2a cells were transfected and treated as described for panel A. The apoptotic index of transfected cells was determined. Triplicate samples were analyzed per data point, and the graph represents three independent experiments. (D) Control (upper panels) or differentiated (lower panels) N2a cells were transfected with GFP and stained with DAPI. Neurofilament (green) was visualized by immunofluorescence with anti-MAP2 antibody. cAMP, cyclic AMP. (E) Differentiated N2a cells were transfected with 0.3 μg of APPct, Fe65, and Tip60 and treated with EOH or E2. The apoptotic index of GFP-positive cells was determined as described for panel C. (F) N2a cells were transfected with 4 μg of plasmids, as indicated. Forty-eight hours later, cells were treated for 45 min, and caspase-3 activity was determined. Triplicate samples were analyzed for each data point, and the data were reproduced. (G) HeLa cells were transfected and treated as described for panel A. The only difference is that 0.3 μg of pLENhERα or a control vector was included in the transfection mix. The number of transfected cells was determined. (H) Cells were transfected as described for panel A and stained with DAPI. Representative micrographs are shown. (I) HeLa cells were transfected and treated as described for panel A. The apoptotic index of transfected cells was determined.