Figure 2
The lysosomal-METRIQ probe detects lysosomal integrity. (A) Detection of several stresses induced by representative inhibitors using flow cytometry. HeLa Tet-On cells expressing the lysosomal-METRIQ probe were cultured with Dox for 48 h and treated with vehicle (DMSO), bafilomycin A1, brefeldin A, or Torin1 for 12 h prior to analysis. The green and red fluorescence intensities were determined by flow cytometry. Representative dot plots of sfGFP versus mCherry fluorescence intensity and corresponding histograms are shown. (B) The red/green fluorescence ratios are shown (n = 4). The fluorescence ratios were based on the high green emission window as defined in (A). The data represent the mean ± SD. The data are representative of at least three independent experiments; *p < 0.05 and **p < 0.01. (C) DNase IIα-sfGFP, but not mCherry, is transported to lysosomes. HeLa cells expressing the lysosomal-METRIQ probe were incubated in medium containing Dox for 48 h and treated with vehicle (DMSO), E64d with pepstatin Aand leupeptin, or Torin1 for 24 h before fixing. Cells were stained with antibodies against LAMP1 and TRAPα, and analysed by immunofluorescence microscopy. A magnified image of the indicated region is shown in the inset. Scale bar, 20 μm. (D) The lysosomal-METRIQ probe can also detect increased lysosomal biogenesis. HeLa cells expressing the probe were cultured with Dox for 48 h and treated with the mTOR inhibitor Torin1 for 12 h prior to flow cytometry. The red/green fluorescence ratios are shown (n = 4). The data represent the mean ± SD; *p < 0.05 and **p < 0.01. (E) Phenotype of the lysosomal-METRIQ probe under lysosomal upregulation.
