Figure 5.

IL-33 overexpression increases the number of mitochondria and decreases mitochondrial autophagy. BMDMs from BALB/c or Il33Tg mice were cultured in fresh medium, with or without IL-4 (25 ng/mL) and rapamycin (Rap) (10 μM). After 24 h of stimulation, the expression of VDAC1, Cytc, Hsp60, Parkin, PINK1, p62, LC3II and I was analyzed by Western blot (A). BMDMs were cultured as described in Figure 6. The number of labeled mitochondria was measured by flow cytometry (B) and fluorescence staining (C) (scale bar: 20 μm). BMDMs were cultured as described in Figure 6. LysoTracker was used to label lysosomes, and MitoTracker was used to label mitochondria to detect the localization of mitochondria and lysosomes (scale bar: 10 μm) (D). After BMDMs were treated as described above and incubated overnight at 4 °C, they were incubated with secondary antibodies with different fluorescent tags for 1 h to detect the localization of VDAC1 and Parkin (scale bar: 10 μm) (E). Data are representative of 3 experiments.