Figure 2. Panel A and B show line graphs... | Rockefeller University Press

Figure 2.

Mercury lamp illumination engages Ca2+ER stores to drive astrocytic Ca2+elevations. (A and B) Inhibition of lamp-evoked Ca2+ oscillations by 2-APB (100 µM, 2 h). DMSO as a control. Data analysis was performed using a paired two-tailed Wilcoxon test, ns, not significant; **P < 0.01. n = 10 independent experiments. Related to Videos 3 and 4. (C) Schematic of the isolation and purification of astrocytes from Itpr1F/FItpr2F/FItpr3F/F mice. (D) Genotyping of P1 Itpr1F/FItpr2F/FItpr3F/F pups. (E and F) Western blot analysis of IP3R protein expression in control and TAT-Cre–treated astrocytes. Data analysis was performed using a two-tailed unpaired t test after normality verification by the Shapiro–Wilk test, **P < 0.01; ***P < 0.001. Error bars represent the mean ± SEM; n = 3 independent experiments. (G) Time series of Ca2+ signals (GCaMP6f, green) in control and IP3R1/2/3 TKO astrocytes before and after mercury lamp illumination. Scale bar: 10 µm. Related to Video 5. (H and I) Quantification of Ca2+ oscillation frequency in control and IP3R1/2/3 TKO astrocytes under resting, and Local and Global illumination protocols. Data analysis was performed using one-way repeated-measures ANOVA; data normality was tested by the Shapiro–Wilk test, ns, not significant; *P < 0.05. n = 6 independent experiments. (J) Ca2+ signals in astrocytes imaged in normal versus Ca2+-free media under Local and Global illumination. Scale bar: 10 µm. (K) Ca2+ event frequency in processes under Local illumination in normal and Ca2+-free media. Data analysis was performed using a two-tailed paired t test after normality verification by the Shapiro–Wilk test, ns, not significant. n = 5 independent experiments. (L) Ca2+ event frequency in soma under Global illumination in normal and Ca2+-free media. Data analysis was performed using a two-tailed paired t test after normality verification by the Shapiro–Wilk test, ns, not significant. n = 5 independent experiments. (M) Effects of 3 mM EGTA, 2 µM thapsigargin, and 9 mW illumination on astrocytic morphology. Scale bars: 10 μm (whole cell); 5 μm (zoom in). Source data are available for this figure: SourceData F2. Refer to the image caption for details.

Panel A and B show line graphs depicting the total Ca2+ positive events per cell under different conditions. The x-axis represents illumination conditions, and the y-axis represents the total Ca2+ positive events per cell. Panel C is a schematic diagram illustrating the isolation and purification of astrocytes from Itpr1F/FItpr2F/FItpr3F/F mice. Panel D shows a gel electrophoresis image for genotyping of P1 Itpr1F/FItpr2F/FItpr3F/F pups. Panel E presents a western blot analysis of IP3R protein expression in control and TAT-Cre-treated astrocytes. Panel F is a bar graph quantifying the relative protein levels of IP3R1, IP3R2, and IP3R3. Panel G contains images of Ca2+ positive signals in control and IP3R1/2/3 TKO astrocytes before and after mercury lamp illumination. Panel H and I are line graphs quantifying the Ca2+ positive oscillation frequency under different illumination protocols. Panel J shows images of Ca2+ positive signals in astrocytes imaged in normal versus Ca2+ positive-free media under Local and Global illumination. Panel K and L are line graphs showing the Ca2+ positive event frequency in processes and soma under different conditions. Panel M contains images depicting the effects of various treatments on astrocytic morphology.

Mercury lamp illumination engages Ca ²⁺ ER stores to drive astrocytic Ca ²⁺ elevations. (A and B) Inhibition of lamp-evoked Ca²⁺ oscillations by 2-APB (100 µM, 2 h). DMSO as a control. Data analysis was performed using a paired two-tailed Wilcoxon test, ns, not significant; **P < 0.01. n = 10 independent experiments. Related to Videos 3 and 4. (C) Schematic of the isolation and purification of astrocytes from Itpr1^F/FItpr2^F/FItpr3^F/F mice. (D) Genotyping of P1 Itpr1^F/FItpr2^F/FItpr3^F/F pups. (E and F) Western blot analysis of IP3R protein expression in control and TAT-Cre–treated astrocytes. Data analysis was performed using a two-tailed unpaired t test after normality verification by the Shapiro–Wilk test, **P < 0.01; ***P < 0.001. Error bars represent the mean ± SEM; n = 3 independent experiments. (G) Time series of Ca²⁺ signals (GCaMP6f, green) in control and IP3R1/2/3 TKO astrocytes before and after mercury lamp illumination. Scale bar: 10 µm. Related to Video 5. (H and I) Quantification of Ca²⁺ oscillation frequency in control and IP3R1/2/3 TKO astrocytes under resting, and Local and Global illumination protocols. Data analysis was performed using one-way repeated-measures ANOVA; data normality was tested by the Shapiro–Wilk test, ns, not significant; *P < 0.05. n = 6 independent experiments. (J) Ca²⁺ signals in astrocytes imaged in normal versus Ca²⁺-free media under Local and Global illumination. Scale bar: 10 µm. (K) Ca²⁺ event frequency in processes under Local illumination in normal and Ca²⁺-free media. Data analysis was performed using a two-tailed paired t test after normality verification by the Shapiro–Wilk test, ns, not significant. n = 5 independent experiments. (L) Ca²⁺ event frequency in soma under Global illumination in normal and Ca²⁺-free media. Data analysis was performed using a two-tailed paired t test after normality verification by the Shapiro–Wilk test, ns, not significant. n = 5 independent experiments. (M) Effects of 3 mM EGTA, 2 µM thapsigargin, and 9 mW illumination on astrocytic morphology. Scale bars: 10 μm (whole cell); 5 μm (zoom in). Source data are available for this figure: SourceData F2.