17-AAG and rapamycin promote the accumulation of LC3-II and the formation of LC3 positive puncta which is inhibited by 3-MA.

(A) Immunoblot analysis of LC3 and HSP70. Cells were treated with 17-AAG (AAG, 50 nM), rapamycin (20 µM), or 3-methyladenine (3-MA, 10 mM) or with a combination of 17-AAG and 3-MA for 7 h and 24 h, respectively. Cell lysates were prepared and immunoblot analysis was carried out with antibodies against the individual proteins as indicated on the right. Co, untreated control. (B) OLN-A53T cells were either untreated (Co, a–c), or treated for 24 h with 50 nM 17AAG (AAG, d–f), or with a combination of 50 nM 17AAG and 10 mM 3-methyladenine (3-MA, g–i), or with 20 µM rapamycin (Rapa, j–l), and then subjected to indirect immunofluorescence staining using antibodies against α-synuclein (SNL-4, a,d,g,j; red) and LC3 (b,e,h,k, green). In (c,f,i,l) the overlays with DAPI staining are shown. Scale bar, 25 µm. (C) Confocal images of cells treated with 17-AAG as in (B, d–f) are shown. Arrow heads indicate colocalization of LC3 staining with α-synuclein. Scale bar, 5 µm.