Dynamin-Related Protein 1 Is Involved in Mitochondrial Damage, Defective Mitophagy, and NLRP3 Inflammasome Activation Induced by MSU Crystals

Figure 3

MSU crystals accelerate the initiation of mitophagy and upregulate the formation of mitolysosomes. (a) The protein levels of PINK1, PRKN, and OPTN were detected by immunoblot in BMDMs primed with LPS (100 ng/ml, 1 h) and treated with MSU crystals (25, 50, 75 μg/ml) at different concentrations for 12 h. (b) Immunoblotting was performed on PINK1, PRKN, OPTN, BECN1, MAP1LC3B-II, and SQSTM1 protein levels in BMDMs primed with LPS (100 ng/ml, 1 h) and treated with MSU crystals (75 μg/ml) for different time points (4 h, 8 h, 12 h). (c) The mt-Keima assay was employed to assess mitophagy in BMDMs primed with LPS (100 ng/ml, 1 h) and treated with MSU crystals (75 μg/ml) for 12 h. The fluorescence intensity ratio of 561/488 nm was used to quantify mitophagy index. Scale bars are 10 μm, experimental replicates with 100 cells analyzed. (d) Western blot analysis and the expression of MAP1LC3B-II and SQSTM1 in BMDMs primed with LPS (100 ng/ml, 1 h) and exposed to MSU crystals (75 μg/ml) for 12 h followed by treatment with 400 nM bafilomycin A1, added during the last 4 h of the 12 h treatment period. Data are shown as . All IB show one representative out of 3. vs. without MSU crystal treatment, ^# vs. MSU crystal treatment.

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