Figure 1
Development of the lysosomal-METRIQ probe, a novel fluorescent construct. (A) Schematic diagrams of the measurement of membrane trafficking and lysosome integrity using fluorescent proteins. (B) Predicted phenotypes of the lysosomal-METRIQ probe against several stresses. (C) sfGFP, but not mCherry, is degraded in lysosomes. Tetracycline-On (Tet-On) HeLa cells expressing sfGFP-mCherry as a negative control or the lysosomal-METRIQ probe containing DNase IIα, ASAH1, or LAMP1 were cultured in the presence of doxycycline (Dox). Following removal of Dox, the cells were incubated for 12 h prior to flow cytometry. Dot plots of GFP versus mCherry fluorescence intensities are shown. (D) Differences in the lysosomal pathway or lysosomal activity under basal conditions among the cell lines. HeLa, HEK293FT, H1299, HT1080, and U2OS Tet-On cells expressing the lysosomal-METRIQ probe were cultured with Dox for 48 h and treated with vehicle (DMSO) or bafilomycin A1 for 12 h prior to flow cytometry (n = 3). The data represent the mean ± standard deviation (SD); **p < 0.01.
