Figure 5

Dysfunction of lysosomes induced by ZnONPs. (a) FACS analysis of cells stained with LysoTracker Red DND-99. The effect of vehicle and ZnONPs (30 μg/ml) treatment on lysosomes amount was analyzed 24 h after treatment. Images are representative of three independent experiments. (b) Western blot analysis of LAMP-1, LAMP-2, cathepsin B and cathepsin D expression levels in A549 cells treated with ZnONPs (30 μg/ml) at an indicated time. β-Actin served as a loading control. Images are representative of three independent experiments. (c) A549 cells were pretreated with DTPA (1 mM), followed by treatment with ZnONPs (30 μg/ml). Western blot analysis of LAMP-1 and LAMP-2 expression levels was performed at 12 or 24 h after treatment. β-Actin served as a loading control. Images are representative of three independent experiments. (d) A549 cells were pretreated with DTPA (1 mM), followed by treatment with ZnONPs (30 μg/ml). FACS analysis of cells stained with LysoTracker Red DND-99 was performed to detect the amount of lysosomes at 24 h after treatment. Images are representative of three independent experiments. (e) A549 cells were transfected with 75 nM siRNA against LAMP-1, LAMP-2 or control siRNA for 48 h. Western blot analysis was performed to verify the knockdown efficiency. Images are representative of three independent experiments (top panel). Then, cells were treated with 30 μg/ml ZnONPs, and MTS analysis was performed to detect the cell viability at 24 h after treatment with 30 μg/ml ZnONPs. Data are representative of three independent experiments (n=6 for each group) and values are expressed in mean±S.E.M. **P<0.01 (bottom panel). (f) The wild-type (WT) A549 cell line and the A549 cell line stably expressing LAMP-2-RFP were determined by immunofluorescence. Note that only LAMP-2-RFP cells represented red signal and had colocalization with LAMP-1, indicating correct expression of LAMP-2-RFP. Images are representative of three independent experiments. Scale bar=10μm (top panel). WT and LAMP-2-RFP cells were treated with ZnONPs at 30, 50 or 100 μg/ml. MTS analysis was performed 24 h after ZnONPs treatment. Data are representative of three independent experiments (n=6 for each group) and values are expressed as mean±S.E.M. *P<0.05 and **P<0.01 (bottom panel)