Fig 1: Gemcitabine promotes lysosomal function.A MIA PaCa-2 cells were incubated for 24 h with vehicle (Control) or gemcitabine (10 µM) before lysosome labeling using anti-LAMP1 antibody. Nuclei were stained with DAPI. Scale bars, 10 µm. Graph shows numbers of LAMP1 puncta per cell calculated using CellProfiler software. Scatter dot plot shows data as means ± SD; n = 31‒48 cells from three independent experiments. Data were statistically analyzed using unpaired t-tests. B Representative images of live MIA PaCa-2 cells incubated with vehicle (Control) or gemcitabine (10 µM) for 24 h then stained with LysoTracker. C Representative images of live MIA PaCa-2 cells incubated with vehicle (Control) or gemcitabine (10 µM) for 24 h then stained with Magic Red. D HeLa cells were incubated for 24 h with vehicle (Control) or gemcitabine (10 µM) before lysosome labeling using anti-LAMP1 antibody. Nuclei were stained with DAPI. Scale bars, 10 µm. Graph shows numbers of LAMP1 puncta per cell calculated using CellProfiler software. Scatter dot plot shows data as means ± SD; n = 22–28 cells from three independent experiments. Data were statistically analyzed using unpaired t-tests. E Representative images of live HeLa cells incubated with vehicle (Control) or gemcitabine (10 µM) for 24 h then stained with Lysotracker. F Representative images of live HeLa cells incubated with vehicle (Control) or gemcitabine (10 µM) for 24 h then stained with Magic Red. G MIA PaCa-2 cells were incubated with vehicle (−) or gemcitabine (10 µM) for the indicated periods. Total cell lysates were analyzed by immunoblotted using anti-CTSB and β-ACTIN antibodies. Levels of pro-CTSB are barely detectable compared with mature CTSB.
Fig 2: Interfering with TFEB expression impairs gemcitabine-induced lysosomal function.A Quantification of MIAshNT and MIAshTFEB cells incubated for 24 h with vehicle (Control) or gemcitabine (Gem; 10 µM) and stained with LysoTracker. Data are shown as means ± SD of n = 471–2196 cells from three independent experiments and were statistically analyzed using two-way ANOVA with Tukey post hoc tests. B Quantification of MIAshNT and MIAshTFEB cells incubated for 24 h with vehicle (Control) or gemcitabine (Gem; 10 µM) and stained with Magic Red. Data are shown as means ± SD of n = 1220–3156 cells from three independent experiments and were statistically analyzed using two-way ANOVA with Tukey post hoc tests. C Activity of CTSB in MIAshNT and MIAshTFEB cells incubated with vehicle (Control) or gemcitabine (Gem; 10 µM) for 24 h (N = 6 from three color-coded independent experiments). Data show color-coded independent experiments, means ± SD and were statistically analyzed using two-way ANOVA with Tukey post hoc tests. D MIAshNT and MIAshTFEB cells were incubated for 48 h with vehicle (Control) or gemcitabine (10 µM). Total cell lysates were analyzed by immunoblotting using anti-TFEB, CTSB, and GAPDH antibodies.
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