Three-dimensional model of mTOR and docking simulation between mTOR and PP242 and MHY1485.

<p>A three-dimensional model of mTOR (NCBI GI number: 4826730) was built using SWISS-MODEL program (A). Homologue structural template used was 1E8X (18% sequence identity and 6.9E-44 e-value). The Z-score of the predicted model was −4.8. We checked the quality of each residue on the mTOR structure model. The internal region, which bound ATP and target compounds, was more reliable (blue) than the external region (red) of mTOR. The estimated residue error was visualized using a color gradient from blue (more reliable regions) to red (potentially unreliable regions, estimated error above 3.5 Å). B shows the docking simulation between mTOR and PP242 and MHY1485. The magenta compound is PP242, which was used as a control compound. The cyan compound is MHY1485, and the yellow compound is ATP. The binding energies of the compounds were −7.28 kcal/mol (PP242) and −7.55 kcal/mol (MHY1485).</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

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

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