Fig. 2. Overexpressed SK1 protects astrocytes during starvation.

a An example of longitudinal imaging of astrocytes. Primary astrocytes were transfected with mApple to visualize their morphology. After transfection, the same group of astrocytes were imaged longitudinally with an automated microscope at different time points. The first image is a montage of non-overlapping images captured in one well of a 24-well plate. Scale bar is 400 µm. The adjacent panels are zoomed in to three cells, to demonstrate longitudinal single-cell tracking. Scale bar is 50 µm. Arrows indicate two astrocytes that died before the last imaging event. b Astrocytes were transfected with mApple and GFP, as control, or with mApple and SK1-GFP, or with mApple and a plasmid that encodes a dominant-negative form of SK1 (dnSK1) tagged to GFP. SK1- or dnSK1-expressing cells were maintained in DMEM supplemented with 10% fetal bovine serum (SK1 or dnSK1) or in Hanks’ balanced salt solution (starvation, starv; SK1 + starv or dnSK1 + starv) and tracked with an automated microscope for 36 h. Risk of death curves demonstrate that SK1 expression protects astrocytes during starvation. ***p (dnSK1 vs dnSK1 + starv) = 0.0001, **p (GFP vs dnSK1) = 0.0253; p (GFP vs SK1) = 0.495, p (SK1 vs SK1 + starv) = 0.2184 (log-rank test). Fifty astrocytes per group were analyzed from three independent experiments. c Primary cortical astrocytes were maintained in basal conditions (control, cont), or in Hanks’ balanced salt solution (starvation, starv) overnight. The levels of S1P were measured by liquid chromatography and mass spectrometry. The bar graph represents relative S1P levels. ***p = 0.0001 (t test). Results were pooled from three independent experiments. d A cohort of astrocytes was transfected with mApple and SK1-GFP. Two cohorts of astrocytes were transfected with mApple and a plasmid that encodes a dominant-negative form of SK1 (dnSK1) tagged to GFP. Astrocytes were maintained in Hanks’ balanced salt solution (SK1 + starv or dnSK1 + starv). A cohort of dnSK1-expressing cells was treated with 1 µM S1P (dnSK1 + starv + S1P). Astrocytes were longitudinally imaged with an automated microscope. The addition of S1P partially restored survival of dnSK1-expressing astrocytes under starvation. ***p (SK1 + starv vs dnSK1 + starv) = 0.0001, **p (dnSK1 + starv vs dnSK1 + starv + S1P) = 0.0287, p (SK1 + starv vs dnSK1 + starv + S1P) = 0.0131 (log-rank test). Fifty astrocytes per group were analyzed from two independent experiments. n.s. not significant