Figure 1
From: Identification of ML-9 as a lysosomotropic agent targeting autophagy and cell death
ML-9 stimulates autophagy by downregulating Akt/mTOR pathway in prostate cancer cells. (a) ML-9 inhibits Akt. To assess Akt phosphorylation, LNCaP cells were treated with full or 30 μM ML-9-containing medium for 12 h. Densitometric quantitation for normalized p-Akt and total Akt relative to Actin is shown. Values represent means±S.E.M. n=3. (b) ML-9 inhibits mTOR. LNCaP cells were incubated in full medium, serum-starved medium or full medium with indicated concentrations of ML-9 for 12 h. The expression levels of p-mTOR, mTOR and beta-Actin were analyzed. Densitometric quantitation for normalized p-mTOR and mTOR relative to Actin is shown. Values represent means±S.E.M. n=3. (c) ML-9 increases LC3-II levels in LNCaP cells in a dose-dependent manner. LNCaP cells were incubated in full medium, serum-starved medium or full medium with indicated concentrations of ML-9 for 6 h. Densitometric quantitation for LC3-II/(LC3-II+LC3-I) ratio is shown. Values represent means±S.E.M. n=3. (d) ML-9 increases LC3-II levels in LNCaP cells in a time-dependent manner. LNCaP cells were untreated or treated with 30 μM ML-9 for 2, 6, 12 and 24 h. Densitometric quantitation for LC3-II/(LC3-II+LC3-I) ratio is shown. Values represent means±S.E.M. n=4. (e) LNCaP cells stably expressing eGFP-LC3 (green) were incubated in full, serum-starved or 30 μM ML-9-containing media for 6 h. Quantitation shown on the right represents means±S.D. GFP-positive puncta per cell (n=50) from three independent experiments. (f) TEM images of LNCaP cells untreated and treated with 30 μM ML-9 for 12 h showing autophagosome (black arrow) and autolysosomes/amphisomes (white arrows). N: nucleus
