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Files in this Data Supplement:
Fig. S1. Temporal expression pattern of XNF-ATc1, XNF-ATc2 and XNF-ATc4 in Xenopus laevis. The expression of XNF-ATc1, c2 and c4 was analyzed by RT-PCR using cDNA of different Xenopus stages and the following primers: XNF-ATc1 (5′-TGCCAAATGTTGACACTTACCG-3′ and 5′-GGGTGATGTAGATGGAGAATGCC-3′), XNF-ATc2 (5′-TTCCTGTCACCTCTCTTCCTCCTC-3′ and 5′-TCGTTCATCTGCTGTTCCAATG-3′), XNF-ATc4 (5′-CCAGATGGAAAGTTACACAATGGGAAG-3′ and 5′-GAGAGGAAGAGGGGAACCTGTG-3′).
Fig. S2. XNF-AT transcriptional activity occurs within the neural tube where XNF-ATc3 is expressed. (A) Xenopus embryos injected at the one-cell stage with 300 pg NF-AT-GFP reporter plasmid were analyzed for GFP fluorescence at gastrula (stage 12; a-c), early neurula (stage 13; d,e), late neurula (stage 19-22; f-k), tadpole (stage 27-32; l-r). The embryo in panels (a-c) was coinjected with lacZ mRNA; lacZ expression was analyzed by in situ hybridization (c). Panel (n) shows a higher magnification of GFP fluorescence in the somites of (m). Panels (a,d,f,h,j,l,o,q) are bright field images of embryos in panels (b,e,g,i,k,m,p,r). White arrows indicate GFP fluorescence. (B) CA XNF-ATc3 increases NF-AT-dependent GFP expression. One-cell stage embryos were coinjected with 50 pg CA XNF-ATc3 and 300 pg NF-AT-GFP reporter plasmid. Embryos were analyzed for GFP fluorescence at late neurula stage (a,b) and tadpole stage (c,d). Panels (a) and (c) are bright field images of embryos in panels (b) and (d). While 42% of the NF-AT-GFP reporter injected embryos showed fluorescence at the neurula stage, coinjection of CA XNF-ATc3 lead to 95% of fluorescent embryos. (C) The NF-AT GFP reporter detects activation and inhibition of NF-AT signaling (a-d). (a) Endogenous NF-AT activity measured by targeted injection of the NF-AT GFP reporter. Embryos from one batch were injected animally, medially or vegetally with 300 pg NF-AT GFP reporter and promoter activity was measured as the number of fluorescent embryos at gastrula (stage 10.5), neurula (stage 18) and tadpole stages (stage 32). (b) The NF-AT GFP reporter is activated by CA XNF-ATc3. Coinjection of 50 pg CA XNF-ATc3 with 300 pg NF-AT GFP reporter strongly increased the number of fluorescent embryos at gastrula and neurula stages compared to the endogenous NF-AT activity of this embryonic batch (a). (c) Inhibition of endogenous NF-AT-induced transcription using DN XNF-ATc3. Embryos were injected with 300 pg NF-AT GFP reporter plasmid (1) and coinjected with 500 pg (2), 1 ng (3) and 2 ng (4) DN XNF-ATc3. The percentage of fluorescent embryos was determined at neurula stages (20-22). (d) Inhibition of NF-AT reporter activity using the L-type calcium channel antagonist Nimodipine and the calcineurin inhibitor CsA. Embryos were injected with 300 pg NF-AT GFP reporter. Embryos were cultured to midblastula transition (MBT) and then transferred to 1/3 MR containing 10 μM Nimodipine or 4 mM CsA. At neurula stages (19-21) the fluorescent embryos were counted.
Fig. S3. WT XNF-ATc3 rescues the inhibition of NF-AT-dependent transcriptional activity and neural CE by CsA. Luciferase assay. One-cell stage embryos were injected with the minimal IL2 promoter in front of the luciferase coding sequence and luciferase activity was measured at the gastrula stage 10.5. To normalize the data and calculate relative luciferase activity (RLU) a renilla luciferase construct using a CMV promoter was coinjected in all conditions. A mixture of AP-1, Calcineurin and WT XNF-ATc3 (AP-1/CaN/NF-AT) was used to activate NF-AT-dependent transcription. If embryos were incubated after injection in 400μM CsA, NF-AT-dependent transcription was dramatically reduced. This reduction could be rescued by coinjection of 125 pg and 250 pg WT XNF-ATc3.
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