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. 2023 Aug 8;8(15):e167694. doi: 10.1172/jci.insight.167694

Figure 8. SRF-VP16 enhances autophagy Poly-GA aggregate clearance.

Figure 8

(A–D) HEK293 cells transfected with a p62-GFP-mCherry construct coexpressed either SRF-VP16ΔMADS (A and B) or SRF-VP16 (C and D). Cells were imaged over 90 minutes, and pictures show the starting time point (0 minutes; A and C) or 60 minutes (B and D). Enhanced autophagy is indicated by a color change from yellow to red. SRF-VP16 enhanced autophagy propagation as indicated by yellow vesicles at t = 0 (C) turning at t = 60 minutes into red (D). In SRF-VP16ΔMADS–expressing cells, the ratio between GFP/mCherry remained constant (A and B). (E–H) HEK293 cells expressing Poly-GA aggregates (green) were stained with lysotracker (red). SRF-VP16 (G and H), but not as much SRF-VP16ΔMADS (E and F), reduced aggregate size and enhanced colocalization of aggregates with lysosomes (yellow in G and H). (I) The GFP/mCherry ratio was decreased in SRF-VP16 compared with SRF-VP16ΔMADS–expressing cells, indicating enhanced autophagic flux by SRF-VP16 (n = 20 cells each; 3 technical replicates). Data show mean ± SEM. (J) The area of Poly-GA aggregates was lower in SRF-VP16 compared with SRF-VP16ΔMADS–expressing cells (n = 40 cells each). Data show mean ± SD. (K) In SRF-VP16–expressing cells, the Poly-GA/lysosome ratio was higher, suggesting more colocalization of aggregates in lysosomes compared with SRF-VP16ΔMADS (n = 40 cells each; 4 technical replicates). Data show mean ± SEM. Statistical testing was performed by 1-tailed t tests. Scale bar: 10 μm.