Fig 1: GAA activates the autophagy pathway through Axl. BV2 cells were (A) treated with GAA at the indicated concentrations for 6 h or (B) treated with GAA (20 µM) for the indicated periods of time, and whole cell proteins were collected for Western blot. (A) GAA-5 vs. DMSO: p = 0.0452; GAA-20 vs. DMSO: p = 0.0034; n = 3. (B) 6 h vs. 0 h: p = 0.0299; 12 h vs. 0 h: p = 0.0073; n = 3. (C–F) BV2 cells were pretreated with 0.1% DMSO or the indicated Axl antagonist (R428 at 5 µM) for 30 min, followed by administration of GAA (20 µM) and Aß42 (2 µM) for 6 h. Whole cell proteins then were collected for Western blot. (D) Aß42 + GAA vs. Aß42: p <0.0001; Aß42 + GAA + R428 vs. Aß42 + GAA: p < 0.0001; n = 3. (E) Aß42 + GAA vs. Aß42: p = 0.028; Aß42 + GAA + R428 vs. Aß42 + GAA: p = 0.0304; n = 3. (F) Aß42 + GAA vs. Aß42: p = 0.0247; Aß42 + GAA + R428 vs. Aß42 + GAA: p = 0.0009; n = 3. (G–H) BV2 cells were pretreated with R428 (5 µM) for 30 min, followed by administration of FITC-Aß42 (1 µM) and GAA (20 µM) for 6 h, and cells were immunostained with LysoTracker Red. Representative images show the co-localization of Aß42 and lysosomes (yellow). Scale bar: 50 µm. Aß42 + GAA vs. Aß42: p = 0.0469; Aß42 + GAA + R428 vs. Aß42 + GAA p = 0.0057; n = 4. (I) BV2 cells were pretreated with the indicated antagonists of Axl (R428 at 5 µM) and Pak1 (IPA-3 at 20 µM), followed by administration of GAA (20 µM) and Aß42 (2 µM) for 24 h. The intracellular Aß42 levels were measured by ELISA. DMSO at 0.1% was used as the control. GAA vs. Control: p = 0.0012; GAA + R428 vs. GAA: p = 0.0151; GAA + IPA vs. GAA: p = 0.0015; n = 3. n is the number of replicates (biological and technical) used for each of the described results. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. indicated control.