Figure 5 | Scientific Reports

Figure 5

From: Blockage of the lysosome-dependent autophagic pathway contributes to complement membrane attack complex-induced podocyte injury in idiopathic membranous nephropathy

Figure 5

Zymosan activation serum (ZAS) blocks autophagic pathway by inhibiting autolysosome formation. Autophagic flux analysis with tandem mRFP-GFP fluorescent-tagged LC3 (tfLC3) plasmid, which showed GFP and mRFP fluorescence signals (yellow) before the autophagosomes fusion with lysosomes, whereas exhibited only the mRFP fluorescence signal (red) after autophagosome and lysosome fusion to become autolysosome due to GFP fluorescence signal (green) quenched in acid environment. After transfection, the cultured podocytes were subjected to heat-inactivated human serum (HIS), rapamycin (RAP, 10 μM), ZAS, or chloroquine (CQ, 10 μM) treatment for different times, fixed, and analyzed by confocal microscopy. The positive control podocytes treated with RAP displayed much more autolysosomes (free red dots) in the cytoplasm compared with the HIS treated control podocytes. However, ZAS induced accumulation of autophagosomes exhibiting increased yellow puncta but almost no free red dots in the cytoplasm from 4 to 36 h. The number of yellow puncta (autophagosomes) was also significantly increased in the presence of CQ. The representative images at 36 h were shown in this study. Arrowheads indicate autophagosomes. Arrows indicate autolysosomes. Scale bar, 10 μm.

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