Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Stimulation of AMPK prevents degeneration of photoreceptors and the retinal pigment epithelium

doi: 10.1073/pnas.1802724115

Figure Lengend Snippet: Metformin prevents light-induced oxidative stress and DNA damage. LD, mice light-damaged in the absence of metformin; MF, mice treated with metformin for 7 d without light damage; MFLD, mice light-damaged following 7 d of metformin pretreatment; Veh, control mice injected with PBS. (A) IHC labeling of 8-OHdG in retinal cross-sections to evaluate the DNA oxidative damage. Similar results were seen in six independent replicates. (Scale bar, 100 μm.) (B) The ratio of NADH to NAD+ was measured enzymatically from one retina per group. n = 3 biological replicates. *P < 0.03. (C) Mitochondrial DNA content was measured in retinas isolated from mice treated with vehicle or metformin for the indicated number of days, mice 6 h after light damage (LD6h), and mice pretreated with metformin for 7 d before light damage (MFLD6h). DNA from mitochondria and the nucleus were measured by qPCR, and the ratio of mitochondrial DNA relative to nuclear DNA is shown. Data were analyzed by one-way ANOVA and Tukey’s post hoc test. n = 4 to 6 biological replicates per group. *P < 0.05. RPE, retinal pigmented epithelium; onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. Error bars = SD.

Article Snippet: pAMPK antibody (2535S; Cell Signaling), SDH-A and mt-Cox-1 (123545; Abcam), β-actin (8226; Abcam), 8-OHdG (MOG-020P; Genox), rabbit opsin red/green (AB5405; EMD Millipore), goat OPN1SW (N-20) (sc-14363; Santa Cruz), mouse cone arrestin (kind gift of W. Clay Smith, University of Florida), LC3B (2775S; Cell Signaling), ATG5-ATG12 (12994P; Cell Signaling), Beclin (3495P; Cell Signaling), and ATG7 (8558P; Cell Signaling) were used as primary antibodies.

Techniques: Injection, Labeling, Isolation