Fpn-GFP expression protects cells against zinc toxicity. (A) BHK 3286-8-8 cells were transfected with a plasmid expressing GFP, Fpn-GFP, or Fpn162V-GFP under the control of the CMV promoter. The day after transfection, cells were exposed to different concentrations of zinc for 24 hours and cell viability assessed by trypan blue exclusion. The data are expressed as percent viable. (B) Cells as in panel A were exposed to 0, 40, and 80μM zinc and the percentage of GFP positive cells were determined over time. The data are expressed as the percentage of GFP-positive cells. (C) Cells as in panel A were harvested at days 1 and 5, cells lysed, and GFP and tubulin levels assessed by Western blot analysis. (D) Cells as in panel A were exposed to different concentrations of zinc to induced MRE-b-galactosidase expression, cells harvested, and b-galactosidase activity measured. The data are expressed as the percentage maximum. (E) BHK 3286-8-8 cells were transfected with pCMV-Fpn-Flag or empty vector, exposed to 70μM zinc for 2 hours, extensively washed, incubated with 1μM FluoZin-3AM for 30 minutes, and extensively washed. FluoZin-3AM fluorescence was detected with a BioTek plate reader fluorimeter at 485/520 nm (excitation/emission) and the data are expressed as the percentage of variation in FluoZin fluorescence (F) over time with the initial FluoZin fluorescence in control and Fpn-Flag expressing cells at zero time (F0). All experiments were performed a minimum of 3 times.