Fig. 1.

A, B Effects of Tre–car on the cytoplasmic pool of labile zinc in PC12 cells. Average intensity values for the FluoZin-3 emission corresponding to the Zn²⁺ content in the cytoplasm for control untreated cells and treated 20 h with Tre, Car, Tre–car or Tre + Car mixture in RPMI1640 complete medium with 1% HS and 0.5% FBS; 20 µM zinc (Magri et al. 2016) or 50 µM membrane-impermeable zinc chelator DPA treatment were used as a positive and negative control, respectively. The effect of Tre–car, Car or Tre + Car mixture treatment analysed by fluorescence images of cells incubated with zinc probe (A) and quantification of fluorescent intensity normalized to the number of cells presented in each field (B) confirms the significantly increased zinc concentration in PC12 cells. As a baseline to exclude cell auto-fluorescence, PC12 cells were treated only with Hoechst33342 without FluoZin-3. Scale bars are 42 μm. All values are mean ± SD of three independent experiments of 6–8 randomly chosen fields. Significant differences between treatments were determined using one-way ANOVA method ^#p ≤ 0.05, ^##p ≤ 0.01, ^###p ≤ 0.001 versus untreated control cells. C Tre, Car, Tre–car or Tre + Car mixture affected ZnT1 expression. Starved PC12 cells were stimulated for 24 h with 5 mM Tre, Car, Tre–car, Tre + Car, 20 µM zinc or 50 µM membrane-impermeable zinc chelator DPA in RPMI11640 complete medium with 1% HS and 0.5% FBS. The expression level of ZnT1 is reported as a ratio to that of GAPDH. Treatment with Car, Tre–car or Tre + Car mixture significantly increased ZnT1 expression. All values are mean ± SD ^#p < 0.01, ^##p < 0.001 versus untreated control cells (samples n = 4, three individual experiments)