Figure 3.
Buffering Nuclear InsP3 Prevents Nuclear Accumulation of NFAT4
(A) Confocal images compare sub-cellular distribution of RFP-tagged InsP3 buffer containing either a nuclear export sequence (IP3-NES; upper panel), InsP3 buffer containing a nuclear localization sequence (IP3-NLS, middle panel), or both (lower panel). DAPI was used to stain the nucleus.
(B) Fluorescence intensities of the InsP3 buffers are compared between the cytoplasmic and nuclear compartments, denoted C and N, respectively. Each bar is the mean of between 9 and 15 cells.
(C) Migration of NFAT1-GFP (denoted N1) and NFAT4-GFP (N4) into the nucleus is compared following stimulation with thapsigargin for 40 min in the presence of the different IP3 buffers (RFP-tagged), indicated on the left.
(D) Cytoplasmic Ca2+ oscillations are compared between cells expressing RFP-tagged IP3-NLS, IP3-NES, or both.
(E) The number of oscillations to LTC4 produced each 200 s bin are compared for the different conditions. Each point is the mean of between 8 and 11 cells.
(F) The amplitude of each Ca2+ oscillation is compared for the conditions shown. Data for (E) and (F) were extracted from experiments as in (D).
(G) Nuclear Ca2+ oscillations are compared for the different conditions indicated.
(H) The number of nuclear Ca2+ oscillations per 200 s bin is shown for each condition. Each point is the mean of between 10 and 26 cells.
(I) The peak amplitude of each nuclear Ca2+ oscillation is compared. Data for (H) and (I) were extracted from experiments as in (G). In (G)–(I), control denotes mock-transfected cells.
(J) Images compare movement of NFAT1-GFP or NFAT4-GFP for the conditions shown. IP3 buffers were tagged with RFP. LTC4 was applied at 160 nM for 40 min and ionomycin (ionom) was 2 μM.
(K) Histogram summarizes data for the various conditions shown. Rest denotes the unstimulated state. N1 and N4 indicate NFAT1-GFP and NFAT4-GFP. Open bars above filled histograms denote the rescue of NFAT movement following stimulation with ionomycin for 20 min. Each bar is the mean from three independent experiments.
In (B), (E), (F), (H), (I), and (K), data are represented as mean ± SEM. See also Figure S3.