Figure 1.
The chemical structure of the chloroquine compound.
Chloroquine diphosphate salt was purchased from Sigma-Aldrich (San Louis, MO, C6628).
Figure 2.
Effects of chloroquine on zinc ion uptake.
A. A2780 cells were plated in a 96-well plate and treated with chloroquine (ChQ) alone or in combination with ZnCl2 at the indicated concentrations for 1 hour. The FluoZin-3 (1 µM) probe was added and the fluorescent signal was measured by excitation at 490/20 nm and emission at 528/38 nm. Data (n = 3, bars, SEM) are presented as a percentage of the highest fluorescence level detected. *, p<0.01, compared to cells without ChQ treatment, using One-way ANOVA followed by Bonferroni analysis. B. A2780 cells were plated in a 12-well plate and treated with ChQ and ZnCl2 at indicated concentrations for 1 hour. After addition of the FluoZin-3 probe, cells were examined under a fluorescent microscope by excitation at 490/20 nm and emission at 528/38 nm. Shown are representative images from three individual experiments.
Figure 3.
Effects of CuCl2, FeCl2, TPEN and Ca-EDTA on chloroquine-induced zinc ion uptake.
A. A2780 cells were plated in a 96-well plate and pretreated with TPEN or Ca-EDTA for 15 min prior to addition of chloroquine (ChQ) and ZnCl2 at the indicated concentrations for 1 hour. B. A2780 cells were plated in a 96-well plate and treated with ChQ alone or in combination with ZnCl2, CuCl2, or FeCl2 at the indicated concentrations for 1 hour. The FluoZin-3 AM (1 µM) probe was then added and fluorescent signal was recorded by excitation at 490/20 nm and emission at 528/38 nm. Data (n = 3, bars, SEM) are presented as a percentage of the highest fluorescence level detected. *, p<0.01, compared with the fluorescence detected in cells without ChQ treatment, using One-way ANOVA followed by Bonferroni analysis.
Figure 4.
Intracellular zinc ion distribution in A2780 cells after chloroquine treatment.
A. A2780 cells were plated in a 6-well plate and treated with chloroquine (ChQ) and ZnCl2 at the indicated concentrations for 1 hour. After addition of the FluoZin-3 and LysoTracker probes, the cells were examined by confocal microscopy (excitation, 490/20 nm, emission, 528/38 nm for FluoZin-3; 555/28 nm, 617/73 nm for LysoTracker, respectively). Shown are representative images from three individual experiments. B. A2780 cells were plated in a 12-well plate and treated with clioquinol (CQ) and ZnCl2 at the indicated concentrations for 1 hour. Confocal images were captured as we previously described [17].
Figure 5.
Effects of chloroquine plus zinc on caspase 3 activation and LC3B-II expression.
A2780 cells were treated with chloroquine (ChQ) or ZnCl2, alone or in combination at the indicated concentrations. Cell lysates were prepared and western blot was performed using primary antibodies against pro-caspase 3, LC3B-II, PARP, and β-actin. Shown are representative blots from three individual experiments.