Figure 4
From: Identification of ML-9 as a lysosomotropic agent targeting autophagy and cell death
ML-9 stimulates autophagy via both Vps34-dependent and -independent mechanisms. (a) ML-9 increase Beclin1 levels in LNCaP cells. LNCaP cells were incubated in full medium, serum-starved medium or full medium with indicated concentrations of ML-9 for 12 h. Densitometric quantitation for normalized Beclin1 relative to Actin is shown. Values represent means±S.E.M. n=3. (b) Wortmannin effectively reduces LC3-II levels induced by ML-9 at low concentrations but is almost ineffective in reducing LC3-II levels induced by ML-9 at high concentrations. LNCaP cells were incubated in full medium, serum-starved medium or full medium with indicated concentrations of ML-9 for 6 h in the absence or presence of 100 nM Wortmannin for the last 1.5 h. Densitometric quantitation for normalized LC3-II relative to Actin is shown. Values represent means±S.E.M. n=3. (c) 3-MA does not preclude ML-9 induced LC3-II accumulation. LNCaP cells were incubated in full medium, serum-starved medium, 20 μM ML-9-containing medium or 30 μM ML-9-containing medium for 6 h. Alternatively, LNCaP cells were pretreated with 5 mM 3-MA for 1 h followed by the 6-h treatment with full medium, serum-starved medium, 20 μM ML-9-containing medium or 30 μM ML-9-containing medium in the continuous presence of 5 mM 3-MA. Densitometric quantitation for normalized LC3-II relative to Actin is shown. Values represent means±S.E.M. n=3. (d) eGFP-LC3 expressing LNCaP cells were treated with full, serum-starved or 30 μM ML-9-containing media for 6 h in the absence or presence of 100 nM Wortmannin. Quantitation shown on the right represents means±S.D. GFP-positive puncta per cell (n=30)
