Figure 6. Activation of the PI3K/AKT/mTOR pathway and increased biomass in RPEΔMT RPE.

(A–D) Immunoblot detects elevated phosphorylation of (A) PI3K (p85^Tyr458), AKT^Ser473, and GSK-3β^Ser9; (B) TSC2^Thr1462; (C) mTOR^Ser2448; and (D) P70SK6^Thr389 and S6^Ser235/236 in pooled RPE cells from pigmented RPEΔMT mice at 14 weeks, compared with that in controls. (E–H) Original magnification, ×200. (E–L) Immunostaining of retinal sections (E–H, K, and L) and RPE flat mounts (I and J) shows increased reactivity of phosphorylated AKT^Ser473 (F, green), mTOR^Ser2448 (H and J, green), and S6^Ser235/236 (L, green) in cre-expressing RPE cells (red/purple) in albino RPEΔMT mice. Phalloidin staining of RPE flat mounts (I and J, white) outlines cell boundaries. Original magnification, ×630. (K and L) Original magnification, ×200. (M) Protein content is increased in RPE cells of RPEΔMT mice (triplicate assays). ^§P < 0.01; ^#P < 0.001. (N and O) Oil red O staining detects a remarkable accumulation of neutral lipid in 14-week-old RPEΔMT RPE (O, purple), compared with that of a control (N). (P) Confocal microscopy of an RPEΔMT flat mount reveals autofluorescent deposits (arrows) visible over a broad spectrum of excitation wavelengths (e.g., 350, 488, and 633 nm). The deposits are initially visible at 350 nm but bleach rapidly, leading to their orange appearance in the merged picture (arrows). The flat mount was stained with anti–β-catenin (green), which highlights RPE cell boundaries at 488 nm. (Q) A higher magnification Z-stack image of the red boxed area in P. Note that autofluorescent deposits are below β-catenin (green) and therefore inside RPE cells. (P and Q) Original magnification, ×200. (R) Electron micrographs of RPEΔMT RPE reveal abundant structures consistent with lipid-containing granules (arrows). Original magnification, ×5000. (S) Funduscopy shows white deposits in a pigmented RPEΔMT mouse. Original magnification, ×50.