Fig. 1
EVs from γ-secretase inhibitor-treated C99 expressing SH-5YSY are enriched in high molecular weight APP-CTFs. a-c EVs were purified from media of vehicle- or D6-treated SH-C99 cells and validated by NTA (a) and electron microscopy (EM) (b) and immunoblot analysis of exosomal markers (c). a-b Both NTA and EM revealed the presence of vesicles having a mean size around 100 nm in both conditions. c Immunoblots showed the presence of the exosomal markers Hsc70, Alix, Flotilin2 and Tsg101 and the absence of the reticular marker Calnexin. c The same immunoblots were used to detect APP-CTFs. Arrows point to distinct high molecular weight (HMW) immunoreactivities revealed with α-APPct. d-e SH-C99 cells were treated or not with D6 and with or without the α-secretase inhibitor GI254023X (Gi), and intracellular APP-CTF levels were analyzed by immunoblot. The same blot is illustrated at low (low) and high (high) exposure. Bars in (e) correspond to the levels of C99 and C83, as normalized to the levels in D6-treated cells. Data represent means ± SEM, ***p < 000.1 as analyzed by the Mann Whitney U-test. f-g APP-CTFs were detected in EVs or cell lysates (cells) using the α-APPct antibody (f) or the N-terminal directed antibody WO-2 (g). Arrows point to HMW immunoreactivities. Note that some of the immunoreactivities revealed with α-APPct (particularly at the red arrow) were not detected with WO-2. h APP-CTFs were detected by immunoblot in EVs or whole lysates (cells) of vehicle- or D6-treated SH-SY5Y-APPswe cells using α-APPct. Arrows point to HMW-APP-CTFs. Low and high correspond to low or high exposures. i-k SH-C99 cells treated or not with D6 and with or without GW4869 (GW) were analyzed for APP-CTF levels by immunoblot using the α-APPct antibody. The same blot is illustrated at low (low) and high (high) exposure. Arrows point to HMW. Bars in (j and k) correspond to the quantification of C99 and C83 (j) or HMW immunoreactivities (k), as normalized to the levels in D6-treated cells. Data represent means ± SEM, *p < 0.05 as analyzed by the Mann Whitney U-test