Figure 6.

Effects of AuNCs on TFEB nucleocytoplasmic distribution and lysosomal activity. (A) U251N cells were treated with vehicle, 1 μM Au₁₅SG₁₃ or 1 μM Au₁₅PEG in DMEM for the times indicated. Panels depict the results for cells incubated for 30 min with vehicle or AuNCs. TFEB was detected by immunocytochemistry, phalloidin stained F-actin, and Hoechst 33342 demarcated nuclei. Whole cell and nuclear fluorescence signals were quantified, and the nuclear/cytoplasmic ratio was calculated. The percentage of cells with elevated nuclear TFEB signals was determined for two independent experiments. For each condition and time point 80 to 101 cells were scored; bars show averages ± SEM. (B) AuNCs increase cathepsin B activity. Cathepsin B activity was measured in U251N cells treated with vehicle, 1 μM Au₁₅SG₁₃ or 1 μM Au₁₅PEG. Cells were incubated with Magic Red as described in the Methods section. Hoechst 33342 identified nuclei. For quantification, Magic Red fluorescence intensity was normalized to the vehicle control. Bars depict average ± SEM for at least two independent experiments. Between 34 and 92 cells were analyzed per condition and time point; **, p<0.01; ***, p<0.001.