Figure 6.

TiO₂ NPs induced mitochondrial proapoptotic factors and subsequent apoptosis in RAW 264.7 cells.

Notes: (A) Mechanism depicting TiO₂ NPs induced BAX activation; (B) RT-PCR gel images of BAX, BIM, and PUMA; (C) Flow cytometry analysis of apoptosis; (D) Quantitative analysis of apoptotic cell death. Cells were incubated with indicated concentrations of TiO₂ NPs for 24 hours. RT-PCR reactions were performed and PCR products were separated by 1% agarose gel electrophoresis and bands were visualized under UVP Biospectrum-600 (Thermo Fisher Scientific, Waltham, MA, USA) after ethidium bromide staining (EtBr, Sigma-Aldrich, St Louis, MO, USA). Housekeeping gene GAPDH was used as loading control. Then, apoptosis was investigated using Muse™ flowcytometric method with Muse™ Annexin V and Dead Cell Kit. Untreated cells were considered as control in the experiments. Data are presented as the mean ± standard error of mean; ***P<0.001 indicates significant difference when tested with ANOVA. Tukey’s test was used for post hoc tests.

Abbreviations: TiO₂, titanium dioxide; GAPDH, glyceraldehyde-3-phosphate; NPs, nanoparticles; RT-PCR, reverse transcription PCR.