Fig. 5. Quantitation of the effects of dominant negative TC10 and Cdc42 proteins on neuritogenesis of PC12 cells. PC12 cells grown on collagen-coated dishes were mock transfected or transfected with T7-epitope-tagged TC10 or HA-epitope-tagged Cdc42 constructs using LipofectAMINE 2000. Six h posttransfection, cells were transferred to medium containing 20% serum, cultured for 18 h, and then transferred to medium containing 1% serum plus 50 ng/ml NGF. Every 24 h thereafter, half the culture medium was removed and replaced with fresh medium plus 50 ng/ml NGF. At 96 h posttransfection, cells were fixed, permeabilized, and stained with anti-T7 antibody or anti-HA antibody and rhodamine-phalloidin. Cells were mounted with Vectashield and viewed with a Zeiss Axiophot fluorescence microscope. For each construct, a total of ~150 cells from multiple transfections were analyzed for neurite outgrowth and morphological changes. Neurites are classified as at least two cell bodies in length, and morphological changes are classified as cellular flattening and the formation of small extensions. In the presence of NGF, nontransfected cells exhibit a high level of neurite outgrowth (53% of the cells) and morphological change (~36% of the cells). The columns represent the percentage of transfected (TC10 31N, Cdc42 17N) or nontransfected cells (None) with neurites (A) or morphological changes (B).