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

Figure 6

Drp1 knockdown alleviated the accumulation of mitochondrial marker protein, mitophagosomes, and mitolysosomes induced by MSU crystals. (a)–(d) BMDMs were transfected with either Drp1 siRNA or Ctrl siRNA for 48 h, then primed with LPS (100 ng/ml, 1 h), and stimulated with MSU suspension (75 μg/ml, 12 h). (a) Western blot was used to detect TOM20 and Cyto C protein levels. (b) BMDMs were subjected to immunofluorescence staining of LC3 (green) and MitoTracker Deep Red (red). (c, d) After BMDMs stably expressing mt-Keima-COX8 plasmid, transfected with Drp1 siRNA or negative control (NC) siRNA for 48 h, primed with LPS (100 ng/ml, 1 h), and then stimulated with MSU crystals (75 μg/ml) for 12 h. (c) Representative LCM images of mitolysosome formation. The mean fluorescence intensity ratio of 561/488 nm was used to quantify mitophagic ratio. Scale bars are 10 μm, experimental replicates with 100 cells analyzed. (d) FCM experiments and analysis were used to analysis mitophagic ratio. (e) Representative western blots showing the expression of MAP1LC3B-II and SQSTM1 in BMDMs transfected with Drp1 siRNA or negative control (NC) siRNA for 48 h, 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 14 h treatment period. Experiments were repeated for at least three times, and data are shown as from 3 independent experiments. All IB show one representative out of 3. vs. without MSU crystal treatment; ^# vs. MSU crystal treatment + vehicle.

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