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. 2020 Jun 10;12(6):1516. doi: 10.3390/cancers12061516

Figure 5.

Figure 5

Pyridinyl imidazole compounds induce the delocalization of mechanistic target of rapamycin (mTOR) kinase from lysosomes in melanoma cells. (A) EGFP-labeled p18/LAMTOR1, endogenous mTOR, and nuclei (DAPI) were visualized by confocal fluorescence microscopy in A375 cells treated with YM201636 (YM; 1 μM), SB202190 (SB202; 15 μM), SB590885 (SB590; 5 μM), BIRB796 (BIRB; 15 µM), Vemurafenib (Vemu; 1 μM), U0126 (10 μM), and Rapamycin (Rapa; 200 nM). Scale bar: 10 µm. The co-occurrence of two fluorescent signals (p18/LAMTOR1-EGFP and endogenous mTOR) was quantified using ImageJ/Fiji. Measurements of three pictures from the same experiment were used for analysis. Three independent experiments showed similar results. (B) A375 cells were transiently transfected with a plasmid construct encoding EGFP-tagged transcription factor EB (TFEB) and treated for 24 h with YM201636 (YM; 1 μM), SB202190 (SB202; 15 μM), and SB590885 (SB590; 5 μM) and the nuclear translocation of TFEB was analyzed using confocal fluorescence microscopy. Scale bar: 20 µm. Colocalization of TFEB and DAPI was quantified using ImageJ/Fiji software (Pearson’s correlation coefficient), and the results were plotted in a graph. Similar results were obtained in three independent experiments.