Research Paper Volume 13, Issue 8 pp 10866—10890

Pharmacologic activation of autophagy without direct mTOR inhibition as a therapeutic strategy for treating dry macular degeneration

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Figure 1.

Identification of autophagy inducers in primary hfRPE culture. (A) Induction of autophagy by analysis of LC3 lipidation (LC3-II/LC3-I ratio). Cultures were exposed to compounds or vehicle (DMSO) for 24 hours. Torin n=8, GSK n=10, FLBZ n=10, D4476 n=8, GW7647 n=11, JNJ n=10, Amiodarone n=8. (B) Induction of autophagy by analysis of LC3 puncta formation (LC3 staining in green) using LC3 Puncta Index described previously [44]. Scale bar: 10 μm. Torin n=7, GSK n=3, FLBZ n=4, D4476 n=7. (C) Autophagy flux assays. After application of inducers or vehicle (DMSO) for 22.5 hours, 25mM of NH4Cl, a lysosomal alkalizing agent, or H2O were added for a final 1.5 hours to inhibit autophagy flux. Resulting increases in the LC3-II/LC3-I ratio indicate that the compound induces autophagy flux. Torin n=6, GSK n=3, FLBZ n=6, D4476 n=5. Uncropped blots for Figure 1 in Supplementary Figure 3. *p < 0.05, **p < 0.01.