Fig. 2. Organellar targeting of DTT-205 and DTT-304.

Human osteosarcoma U2OS cells stably expressing the nuclear marker histone H2B together with red fluorescent protein (RFP), the ER marker calreticulin (CALR) labeled with green fluorescent protein (GFP), galactose-1-phosphate uridylyltransferase GALT1, a marker of the Golgi apparatus fused to GFP, DIABLO co-expressing GFP as an indicator for mitochondria and LAMP1-GFP as a lysosomal marker were treated with 1.25 µM Pacific blue-labeled DTT peptides in the presence or absence of lysosomal acidification that was blocked or not with bafilomycin A1 (BAFA1). Both DTT-205 and DTT-304 accumulated in lysosomal structures, an effect that was inhibited with BAFA1. Representative images of confocal assessment (a, b; size bar equals 5 µm) and relative cooccurrence of Pacific Blue label with organellar markers was assessed (c, d; mean ± SEM of a minimum of five view fields). Wild-type U2OS cells were stained with LysoTracker green and the decrease in lysosomal content was assessed upon treatment 0.65 or 1.25 µM DTT peptides for 6 h by epifluorescence microscopy. Representative images (e) and quantifications (f, g) are depicted (size bar equals 10 µm; mean ± SD of triplicate assessments, Student’s t test, *p < 0.5). Viability was measured in living cells by means of the exclusion dye propidium iodide 6 h post treatment with 0.65 to 10 µM DTT-205 (h) and DTT-304 (i) in the presence or absence of BAFA1 by live cell microscopy (mean ± SD of triplicate assessments, Student’s t test, *p < 0.5, **p < 0.01, ***p < 0.001). BAFA1 partially decreased the cytotoxic effect of the DTT peptides