FIGURE 4.

Palmitate decreases lysosomal acidification and cathepsin L activity. INS1 cells were stained with either the probe for acidic vesicular organelles LysoTracker red, or the pH indicator LysoSensor yellow/blue, or the cathepsin L activity indicator magic red after treatment with or without palmitate for 14 h. a, flow cytometry intensity histogram of cells stained with LysoTracker red. b, confocal microscopy analysis of INS1 cells expressing GFP-LC3 (green) and stained with LysoTracker red (red). Nuclei were co-stained with Hoechst (purple). Bar, 10 μm. Quantification of the number of AVOs. Error bars, S.D., *, p < 0.05. Note that LysoTracker red and GFP-LC3 did not co-localize, reflecting the sensitivity of GFP to the acidic pH of the lysosomes and autolysosomes. c, palmitate decreases AVO acidification. Images show INS1 cells stained with LysoSensor yellow/blue and imaged using two filter ranges, 404–456 nm (blue) and 510–641 nm (yellow). Subcellular organelles with lower pH are identified as pixels with increased yellow/blue ratio. Error bars, S.E., n = 7. d, average AVO size measured from LysoSensor images. e, palmitate suppresses cathepsin L activity. Staining for MR-cathepsin L. The graph expresses the average red intensity. Error bars, S.E., n = 6, **, p < 0.01. f, pHrodo-dextran fluorescence intensity as a measure for endosome acidity. After 14 h in the presence or absence of palmitate, the cells were incubated with pHrodo-dextran for 1 h and pHrodo intensity was measured; Error bars, S.E., n = 3, **, p < 0.01.