Increase of the LC3II/LC3I ratio and LC3II-positive vacuoles by MHY1485.

<p>Western blot analysis was performed to detect LC3II and LC3I. Ac2F cells were treated with different concentrations of MHY1485 and rapamycin 5 µM as a positive control for 6 h. Bars represent the LC3II/LC3I ratio calculated by normalizing the LC3II/LC3I ratio from MHY1485-treated or rapamycin-treated samples with the LC3II/LC3I ratios from untreated samples (A). Control cells treated with same volume of vehicle or MHY1485 (2 µM) for 1, 6 or 12 h were collected. Bars represent the LC3II/LC3I ratio calculated by normalizing the LC3II/LC3I ratio from MHY1485-treated samples with LC3II/LC3I ratio from control samples at 1 hour (B). β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05, **p<0.01, ***p<0.001; n = 3). Live-cell confocal microscopic images of AdGFP-LC3-transfected Ac2F cells treated with 2 µM MHY1485 for 1, 6 or 12 h are shown. The images show the GFP-LC3-positive vacuoles (upper, green), corresponding phase contrast images (middle) and merged images (bottom). Scale bar, 20 µm.</p

Activation of mTOR by MHY1485.

<p>Western blot analysis was performed to detect the change of total protein level and levels of phosphorylated forms of mTOR and 4E-BP1 reflecting the activity of mTOR. Ac2F cells were treated with MHY1485 of different concentrations and rapamycin 5 µM as a positive control for 1 h. Bars represent the phospho-mTOR(Ser2448)/mTOR ratio and the phospho-4E-BP1(Thr37/46)/4E-BP1 ratio normalized with the ratio from untreated samples, respectively. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05; n = 3).</p

MHY1485 inhibition of starvation-induced colocalization between autophagosomes and lysosomes.

<p>Live-cell confocal microscopic images of AdGFP-LC3 (green) and lysosomes (red) stained by LysoTracker were collected from Ac2F cells under starvation or co-treatment of 2 µM MHY1485 for 6 h. Merged images show the co-localization of LC3-positive autophagosomes and lysosomes (yellow). Scale bar, 20 um. Bars represent the level of overlap index.</p

Brief scheme of the synthetic process of MHY1485.

<p>Brief scheme of the synthetic process of MHY1485.</p

Failure of the increase of autophagic flux.

<p>Western blot analysis was performed with samples from cells treated in 2 µM MHY1485 for 6 h. The lysosomotropic agents bafilomycin A1 (bafA1, 10 nM) and chloroquine (100 µM) were applied 1 h before the cell harvest to measure the autophagic flux (A, B). Bars represent the LC3II/LC3I ratio normalized with the LC3II/LC3I ratio from untreated samples. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (**p<0.01; n = 3). C and D show the immunoblots of p62 and beclin-1 in samples from the cells treated with MHY1485 or the same amount of vehicle for different times. The blots were quantified by densitometry and were normalized with the control sample at 1 h expressed as mean±SD (*p<0.05; n = 3).</p

Inhibition of starvation-induced autophagic flux by MHY1485.

<p>Western blot analysis was performed to measure autophagic flux. Ac2F cells under starvation or co-treatment of 2 µM MHY1485 for 6 h were treated 1 h before the cell harvest with lysosomotropic agents bafilomycin A1 (10 nM) and chloroquine (100 µM) (A, B). Bars represent the LC3II/LC3I ratio normalized with the LC3II/LC3I ratio from untreated samples. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05, ***p<0.001; n = 3).</p