Nano rare-earth oxides induced size-dependent vacuolization: an independent pathway from autophagy

Figures & data

Figure 1 Massive vacuolization of GFP-LC3/HeLa cells induced by Y₂O₃, Yb₂O₃ but not La₂O₃. A) Direct observation of vacuolization and GFP-LC3 dot aggregation via fluorescent microscopy (treated with 50 μg/mL oxide particles, 24 h). B) The vacuoles in GFP-LC3/HeLa cells treated with Yb₂O₃ (50 μg/mL, 24 h). C) GFP-LC3 aggregation dose effect after treatment with Yb₂O₃ for 24 h. D) GFP-LC3 aggregation time curve after treatment with 50 μg/mL Yb₂O₃. E) Vacuolization percentage dose effect in GFP-LC3/HeLa cells after treatment with Yb₂O₃ for 24 h. F) Vacuolization percentage time curve essay after treatment with 100 μg/mL Yb₂O₃. Mean ± SEM; n = 3.

Notes: *P < 0.05; **P < 0.01.

Figure 2 The vacuolization of GFP-LC3/HeLa cells, induced by Y₂O₃, Yb₂O₃, is coupled with a typical autophagic process. A) Ultrastructural features of autophagosomes treated with PBS or 50 μg/mL of Yb₂O₃ for 2 days, as revealed by TEM. Right panels are high magnification images of left panels. Typical AVs (black arrows) are indicated, b–d. Autophagic marker dye staining assay on GFP-LC3/HeLa cells treated with 50 μg/mL Yb₂O₃ for 24 h. B) 10MM MDC, 15 min. C) 75 nM LysoTracker Red (LT), 15 min. D) 20 nM MitoTracker Red (MT), 15 min. Hoechst 33342 (HO) dye was used to show nucleus subcellular location. E) LC3 protein type conversion and the accumulation of LC3-ll in 48 h, Cells were treated with 50 μg/mL Yb₂O₃ and then subject to the western blotting. GAPDH was loading control. F) P62 protein degradation rate in autophagy pathway was measured by western blotting. Cells were treated with 50 μg/mL Yb₂O₃ for 24 h and then analyzed. GAPDH was loading control.

Figure 3 The size-dependent effect of vacuolization induced by Y₂O₃. A) GFP-LC3/HeLa cell lines treated with Y₂O₃ nanoparticles no longer contain visible vacuoles. B, C) DLS and TEM picture showed smaller size of nano-Y₂O₃ powder. D, E, F) The supernatant of Y₂O₃ after centrifugation at 1600 rpm also showed similar results.

Figure 4 The size-dependent effect of vacuolization induced by Yb₂O₃. A) sonicated Yb₂O₃ no longer causes visible vacuoles in GFP-LC3/HeLa cell lines. B, C) DLS data and TEM picture showed smaller size of sonicated Yb₂O₃ compared to untreated Yb₂O₃ particles. D) Autophagic marker dye staining assay on GFP-LC3/HeLa cells treated with 50 μg/mL Yb₂O₃ (sonicated) for 24 h. E) LC3 protein type conversion. Cells were either untreated (cont) or treated with 50 μg/mL oxide particles for 24 h and collected for western blotting.

Figure 5 Massive vacuolization induced by Y₂O₃, Yb₂O₃ is an independent event. A) the autophagy blocking effect of Atg5^−/− MEF cells by western blotting. Cells were either untreated (cont) or treated with trehalose 100 μM, rapamycin 50 μg/mL (Tre, Rap, two well-known autophagy inducers as positive control), 50 μg/mL Y₂O₃, Yb₂O₃, I-MEF = immortalized wildtype MEF cells, K-MEF = Atg5^−/− MEF cells. B) Direct vacuolization of K-MEF cells observed under phase contrast microscopy. C, D) 3-MA, the autophagy inducer, does not interfere with vacuolization. Cells were treated with nothing (cont), 50 μg/mL Yb₂O₃, 50 g/mL Yb₂O₃ + 5 μM 3-MA. After observation under fluorescent microscopy, cells were trypsinized, collected and subject to western blotting.