Figure 5
Figure 5. CD40 stimulation sensitizes CLL cells to GA101-induced cell death via a lysosome-dependent mechanism. (A) Confocal microscopy of LysoTracker-labeled unstimulated (bottom panel) and CD40-stimulated CLL cells (top panel) after different time points of treatment with GA101. Shown is the distribution of lysosomes in individual cells visualized in glow-over-under (right: glow-over-under color scale with the highest intensity in blue). Note the increase in both number and activity of lysosomes in CD40-stimulated CLL cells (3T40L medium) compared with unstimulated cells (3T3 medium). After 1 hour of incubation with GA101, CD40-stimulated CLL cells showed clustering of cells (arrow). After 2 hours of incubation with GA101, an increase in LysoTracker signal (blue) was observed in CD40-stimulated CLL cells (top panel), whereas no increase was observed in unstimulated CLL cells. Data shown are representative of 3 individual experiments, each with > 100 cells and identical settings for imaging. Scale bar indicates 5 μm. (B) CD40-stimulated and unstimulated CLL cells were labeled with LysoTracker and analyzed by flow cytometry. Left: representative FACS plot of LysoTracker signal in unstimulated (gray line) and CD40-stimulated CLL cells (black line). Thin black line: unstained control. Middle mean fluorescence intensity (MFI) of LysoTracker in unstimulated and CD40-stimulated CLL cells. Averaged results from 5 CLL patients (mean ± SEM) ***.001 < P < .01 significant difference from control (3T3). Right: Quantification of the number of lysosomes per cell in 3T3, 3T40L, 3T40L + GA 2-hour and 3T40L + GA 6-hour–stimulated CLL cells. Averaged results from 2 independent observers (mean ± SEM) **.001 < P < .01; ***P < .001. (C) Top: Representative Western blot of the 43-kDa procathepsin B (CTSB) of cell lysates from 3T3 and 3T40L CLL cells for 4, 24, or 48 hours. The asterisk (*) marks the position of an aspecific protein band reacting with the antiserum. Tubulin was used as loading control on the same gel. Blot is representative of 4 mutated patients tested. Contrast and brightness were adjusted for optimization. Bottom: in vitro labeling of active GBA in unstimulated and CD40-stimulated CLL cells for different time points (3 different patients). Tubulin was used as loading control on the same samples, run in parallel on a standard SDS-PAGE gel and followed by Western blot. (D) CLL cells were stimulated with 3T3 or 3T40L in a time course (4, 24, and 48 hours), after which time LysoTracker signal was analyzed by flow cytometry. Averaged results from 3 CLL patients are presented as Geomean of LysoTracker signal (mean ± SEM) *.01 < P < .05. (E) CD40-stimulated CLL cells were incubated with RXL or GA101 in the presence or absence of NAC. After 24 hours, cell death was analyzed by measuring MitoTracker signal by flow cytometry. NAC significantly blocked RXL-induced cell death of CD40-stimulated CLL cells, but not GA101-induced cell death. Data are presented as percentage specific cell death (mean ± SEM). Averaged results from 10 independent experiments are shown. *.01 < P < .05. (F) Confocal microscopy of LysoTracker-labeled, CD40-stimulated CLL cells after 2 hours of treatment with GA101 or RXL. Shown is the distribution of lysosomes in individual cells visualized in glow-over-under. GA101 induces an increase in number and activity of lysosomes in contrast to RXL. Scale bar indicates 5 μm. Bar graph shows LysoTracker signal analyzed by flow cytometry. (G) Confocal microscopy of LysoTracker-labeled, CD40-stimulated CLL cells after 2 hours of treatment with GA101 (GA) in the presence or absence of Q-VD, cytoD, or concA. Note that HA and increase in LysoTracker signal were not blocked by Q-VD. An increase in LysoTracker signal was observed in the presence of cytoD, despite the absence of HA. ConcA blocked the GA101-induced increase in LysoTracker signal, but not HA. Scale bar indicates 5 μm.

CD40 stimulation sensitizes CLL cells to GA101-induced cell death via a lysosome-dependent mechanism. (A) Confocal microscopy of LysoTracker-labeled unstimulated (bottom panel) and CD40-stimulated CLL cells (top panel) after different time points of treatment with GA101. Shown is the distribution of lysosomes in individual cells visualized in glow-over-under (right: glow-over-under color scale with the highest intensity in blue). Note the increase in both number and activity of lysosomes in CD40-stimulated CLL cells (3T40L medium) compared with unstimulated cells (3T3 medium). After 1 hour of incubation with GA101, CD40-stimulated CLL cells showed clustering of cells (arrow). After 2 hours of incubation with GA101, an increase in LysoTracker signal (blue) was observed in CD40-stimulated CLL cells (top panel), whereas no increase was observed in unstimulated CLL cells. Data shown are representative of 3 individual experiments, each with > 100 cells and identical settings for imaging. Scale bar indicates 5 μm. (B) CD40-stimulated and unstimulated CLL cells were labeled with LysoTracker and analyzed by flow cytometry. Left: representative FACS plot of LysoTracker signal in unstimulated (gray line) and CD40-stimulated CLL cells (black line). Thin black line: unstained control. Middle mean fluorescence intensity (MFI) of LysoTracker in unstimulated and CD40-stimulated CLL cells. Averaged results from 5 CLL patients (mean ± SEM) ***.001 < P < .01 significant difference from control (3T3). Right: Quantification of the number of lysosomes per cell in 3T3, 3T40L, 3T40L + GA 2-hour and 3T40L + GA 6-hour–stimulated CLL cells. Averaged results from 2 independent observers (mean ± SEM) **.001 < P < .01; ***P < .001. (C) Top: Representative Western blot of the 43-kDa procathepsin B (CTSB) of cell lysates from 3T3 and 3T40L CLL cells for 4, 24, or 48 hours. The asterisk (*) marks the position of an aspecific protein band reacting with the antiserum. Tubulin was used as loading control on the same gel. Blot is representative of 4 mutated patients tested. Contrast and brightness were adjusted for optimization. Bottom: in vitro labeling of active GBA in unstimulated and CD40-stimulated CLL cells for different time points (3 different patients). Tubulin was used as loading control on the same samples, run in parallel on a standard SDS-PAGE gel and followed by Western blot. (D) CLL cells were stimulated with 3T3 or 3T40L in a time course (4, 24, and 48 hours), after which time LysoTracker signal was analyzed by flow cytometry. Averaged results from 3 CLL patients are presented as Geomean of LysoTracker signal (mean ± SEM) *.01 < P < .05. (E) CD40-stimulated CLL cells were incubated with RXL or GA101 in the presence or absence of NAC. After 24 hours, cell death was analyzed by measuring MitoTracker signal by flow cytometry. NAC significantly blocked RXL-induced cell death of CD40-stimulated CLL cells, but not GA101-induced cell death. Data are presented as percentage specific cell death (mean ± SEM). Averaged results from 10 independent experiments are shown. *.01 < P < .05. (F) Confocal microscopy of LysoTracker-labeled, CD40-stimulated CLL cells after 2 hours of treatment with GA101 or RXL. Shown is the distribution of lysosomes in individual cells visualized in glow-over-under. GA101 induces an increase in number and activity of lysosomes in contrast to RXL. Scale bar indicates 5 μm. Bar graph shows LysoTracker signal analyzed by flow cytometry. (G) Confocal microscopy of LysoTracker-labeled, CD40-stimulated CLL cells after 2 hours of treatment with GA101 (GA) in the presence or absence of Q-VD, cytoD, or concA. Note that HA and increase in LysoTracker signal were not blocked by Q-VD. An increase in LysoTracker signal was observed in the presence of cytoD, despite the absence of HA. ConcA blocked the GA101-induced increase in LysoTracker signal, but not HA. Scale bar indicates 5 μm.

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