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. 2019 Jan 29;11:9. doi: 10.3389/fnagi.2019.00009

Figure 4.

Figure 4

Depletion of chaperones promotes cytosolic stress, protein misfolding and is associated with an induction of autophagy and diminished ubiquitin proteasome system (UPS) capacity. (A,B) siRNA treated HEK293 cells expressing FlucDM-GFP were subjected to live-cell imaging. Depicted are representative images 48 h post siRNA transfection (A). As control, cells were treated with 20 μM MG-132 for 6 h (B). Red triangles point out FlucDM-GFP aggregates. Scale bars: 10 μm. (C) Aggregation analysis of the images acquired at 0, 8, 24 and 48 h of (A,B). The green color represents no or a low number of cells with aggregates, an intermediate number of cells with aggregates is indicated in yellow and the red color indicates a high number of cells with aggregates. (D,E) FlucDM-GFP activity 24 and 48 h after siRNA transfection. Relative luciferase activity of the chaperone knockdown conditions and the respective time point (A). As control, cells were treated with 20 μM MG-132 for 6 h (E). Error bars represent the SD of three independent experiments. (F) LC3-II levels of siRNA treated HEK293 cells. Depicted are normalized LC3-II levels. (G) UbG76V-GFP levels of siRNA treated HEK293 cells. (H) Chymotrypsin-like proteasome activity of siRNA treated HEK293 cells.