Figure 1.

Glucose and membrane potential-dependent activation of NFATc3. A) Image of MIN6 cells transiently transfected with mCherry-NFATc3 at low glucose and 30 min after the addition of high glucose. B) As in A for mouse islets virally infected with mCherry-NFATc3. C) As in A for human islets from a healthy donor, virally infected with mCherry-NFATc3. D) Time-course of FRET ratio from D3cpV Ca²⁺ FRET sensor transfected into MIN6. Ca²⁺ changes were measured under four separate conditions: 2 mM glucose (red), 20 mM glucose (blue), 11 mM glucose + diazoxide (green), and 11 mM glucose + FK506 (black). E) NFATc3 translocation to the nucleus in MIN6 cells, for the conditions indicated in D, as measured by the ratio of nuclear to cytoplasmic fluorescence. Traces represent average of all experiments performed in MIN6 cells. F) Mean nuclear-cytoplasmic mCherry-NFATc3 ratio 30 min after treatment in MIN6 cells. G) As in F for primary mouse islets. H) As in F for primary human islets from healthy donors. I) Image of insulin or glucagon staining (green) plus mCherry-NFATc3 (red) in mouse islets following incubation with low (2 mM) or high (11 mM) glucose. J) Mean nuclear-cytoplasmic mCherry-NFATc3 ratio 30 min after incubation in insulin + cells of mouse islets. K) As in J for glucagon + cells of mouse islets. L) As in j for insulin + cells of human islets. M) As in K for glucagon + cells of human islets. Statistical analysis was done using ANOVA with Tukey HSD post hoc test (F–H) or Student's t-test (J–M). ∗, ∗∗, and ∗∗∗ represent p = 0.05, p = 0.005, p = 0.0005, respectively. Data in F averaged over n = 5,11,8,4 plates respectively (18,31,21,8 cells); data in G averaged over n = 3 mice (12,11,8,9 islets); data in H averaged over n = 6 donors (n = 8 for 2G, 11G + FK506; 28,21,24,33 islets); data in J,K averaged over n = 3 mice, data in L,M averaged over n = 3 donors.