Transfection of ATDC5 cells with wild type and mutant Ank. (a) Confocal microscopy of ATDC5 cells transiently transfected with M48T mutant cDNA. Left panel is phase-contrast image of right panel. All transfected cells showed a similar pattern of plasma cell membrane staining, indicating that even mutant Ank molecules were able to translocate to the plasma cell membrane. (b) ELISA assay results showing comparative levels of Ank protein expression in ATDC5 cell lysates versus lysates of independent clones of ATDC5 cells transduced with various ank constructs. All cells express endogenous Ank protein, but transduced cells also express protein derived from expression of transduced constructs. Data represent quadruplicate assays and are representative of results obtained from other independent clones for each transductant. *P ≤ 0.05. (c) WST-1 proliferation assay at day 7 of culture in ATDC5 cells transduced with empty vector and with various wild-type (WT) or mutant ank constructs. At days 7, 14, and 21, 7.5 μl WST-1 reagent was added directly to 150 μl cell medium and incubated for 1.5 hours at 37°C in 5% CO2. Results at all time points consistently exhibited no significant differences in the proliferation of mutant-transduced cells compared with untransduced cells or cells transduced with empty vector. n = 3.