Fig 1. The effects of iron, copper, and HU on cells expressing the anti-apoptotic sequence 14-3-3.

(A) Spot assays were used to assess the ability of wild-type cells expressing 14-3-3, and TC-1 to grow when challenged with 2mM copper (CuSO₄) or 6mM iron (FeCl₃). (B) The viability of cells containing empty vector and 14-3-3 expressing strains untreated (-) or treated (+) with 1.4mM copper (CuSO₄) or 6mM iron (FeCl₃) was determined by examining cells stained with trypan blue. Viability is shown as the mean percentage (%) of the cells that survived after treatment in triplicate experiments that were repeated at least 3 times.*; indicates significant differences between control cells (Vector) cells expressing 14-3-3 that were treated with copper or iron (p <0.001). **; indicates significant differences between control cells (not treated) and cells treated with copper or iron (p <0.001). (C) Spot assay was used to examine cell growth ability of the wild-type cells expressing 14-3-3 on nutrient agar containing 200nM rapamycin (Rap) or 20mM zinc chloride (ZnCl₂). (D) Spot assay using nutrient agar containing 200mM Hydroxyurea (HU) was used to assess the growth of cells expressing 14-3-3. (E) Yeast cells harboring empty vector, as well as vector expressing 14-3-3, TC-1 and YCA1 were serially diluted and spotted on YNB media with galactose alone (Control) or with 1.8mM copper (CuSO₄) or with 5mM iron (FeCl₃). The cells were incubated at 30°C to grow for 3 days.