Figure 1.

Polystyrene (PS) nanoplastic uptake via endocytosis. (A) Mouse embryonic fibroblasts (MEFs) were treated with 100 nm PS-YG (yellow-green) nanoplastics at 200 mg/L for 0 to 48 h. Cells were subjected to immunofluorescence analysis using an anti-α-tubulin (α-Tub) antibody and DNA was visualized with DAPI. Direct fluorescence from PS-YG nanoplastics was also visualized. (B) Endocytosis was blocked using sodium azide. MEFs were pretreated with sodium azide (+ Endocytosis block) or vehicle (− Endocytosis block) for 16 h before 100 nm PS-YG treatment. After 16 h, MEFs were treated with 100 nm PS-YG nanoplastics at the indicated concentration for 24 h. (C) Quantitative analysis of PS-YG fluorescence levels in (A, upper panel) and (B, lower panel). Green fluorescence levels in all α-Tub-positive cells in the field were determined and expressed as the means ± SEM from the number of cells shown. (D) Immunoblot detection (upper panel) and qRT-PCR analysis (lower panel) of Eea1 in MEFs treated with PS-YG at the indicated concentration for 1 day with or without sodium azide pretreatment to block endocytosis. α-Tubulin (α-Tub) was used as a loading control for immunoblot analysis. mRNA expression levels of Eea1 were determined by qRT-PCR (n = 3 each), normalized against Gapdh levels and expressed as the fold change relative to untreated control levels. Representative images of cells or immunoblots are shown. qRT-PCR data are expressed as the means ± SEM from the indicated number of samples. * p < 0.05; ** p < 0.01; *** p < 0.001 between two groups, as indicated by horizontal bars. ND, not determined. NS, not significant. Scale bar, 100 μm.