Fig. 2

Cilostazol (PDE3 inhibitor) increases lysosomal free Zn²⁺ levels, re-acidifies lysosomes, and promotes autophagy flux. a. Fluorescence photomicrographs of FluoZin3-loaded, cultured astrocytes before (left) and after a 1-h treatment (right) with 10 μM cilostazol alone or cilostazol plus the PKA inhibitor H-89 (10 μM) or Zn²⁺ chelator TPEN (500 nM). Cilostazol treatment raised free Zn²⁺ levels in lysosomes, an effect that was blocked by H-89 or TPEN. b. Fluorescence photomicrographs of astrocytes loaded with DND189 (a pH-sensitive lysosomal dye) before (left) and after a 1-h treatment (right) with 100 nM bafilomycin A1 (BA) alone, BA plus 10 μM cilostazol, BA plus cilostazol and PKA inhibitor (H-89, 10 μM), or BA plus cilostazol and TPEN (500 nM). c. Fluorescence images of H4 cells transfected with both GFP-LC3 and RFP-LC3 obtained after a 6-h treatment with 100 nM BA alone, BA plus 10 μM cilostazol, cilostazol alone, or sham washed (CTL). With BA treatment, GFP fluorescence (left) did not disappear, resulting in many yellow spots in the merged image. Addition of cilostazol substantially reduced GFP signals, resulting in a reduction in yellow spots in the merged image. d. Western blots (upper) for p62, a marker of autophagy flux, and corresponding β-actin in samples obtained from astrocytes after a 6-h treatment with 100 nM BA alone, BA plus cilostazol, BA plus PKA inhibitor (H-89, 10 μM), or sham washed (CTL). Another set of Western blots (lower) for p62 and corresponding β-actin in samples obtained from astrocytes after a 6-h treatment with BA alone, BA plus cilostazol, BA plus TPEN, or sham washed (CTL)