Enforced expression of RASSF1A in ACC cells. (A) SW-13 ACC cells that do not express RASSF1A (a) were transfected with empty (a &e), RASSF1A (b - d) or RASSF1A/A13 3S (f - h) expression vectors. Expression of RASSF1A was determined by immunofluorescence detection of RASSF1A protein (b &f) or DDK tag (c &g) using anti-RASSF1 goat polyclonal and anti-DDK mAb respectively followed by anti-goat-FITC and anti-moue-TR secondary antibodies and DAPI for nuclear staining. Note co-localization of RASSF1A and DDK antigens (d &h) and absence of both in a &e. (B &C) Transient transfection was carried out in 6-well plates with a starting density of 80,000 cells/well and allowed to grow for 6 days to test the effect of RASSF1A and RASSF1A/A133S expression on growth potential (B) and survival (C) of SW-13 cells. Graphs represent one of 3 independent experiments that yielded similar results.