Panel A shows fluorescence images of astrocytes co-transfected with GCaMP6f and LAMP1-mCherry under different conditions: resting, DMSO, W7, SMIFH2, and global illumination. The images depict the motility of late endosomes/lysosomes. Panel B is a violin plot showing the mean velocity of LAMP1-positive late endosomes/lysosomes in the DMSO condition. The y-axis represents mean velocity in micrometers per second. Panel C is a violin plot showing the mean velocity in the W7 condition. Panel D is a violin plot showing the mean velocity in the SMIFH2 condition. Panels B, C, and D include statistical annotations indicating significance levels. Panel E shows fluorescence images of astrocytes expressing the ATP sensor GRABATP1.0 under resting, global illumination, and 100 micromolar ATP conditions.
Exploring Ca 2+ -sensitive pathways using inhibitors. (A) Astrocytes were cotransfected with GCaMP6f and LAMP1-mCherry. Late endosomes/lysosome motility following Global illumination was analyzed using the ImageJ TrackMate plugin in astrocytes pretreated for 5 min with DMSO (control), 100 µM W7, or 100 µM SMIFH2. Scale bar: 10 μm. (B–D) Quantification of the mean velocity of LAMP1-positive late endosomes/lysosomes in DMSO, W7, and SMIFH2 conditions. Data analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test, ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001. Error bars represent the mean ± SEM; n = 3 independent experiments. (E) Astrocytes expressing the ATP sensor GRABATP1.0 (green) were imaged before and after stimulation with either 100 μM ATP or Global illumination. Scale bar: 5 μm.