Supporting InformationMuller et al. 10.1073/pnas.0813296106SI TextSI Results. The survival disadvantage of mice globally expressing hyper-activable NFAT1 is not due to deleterious effects of Cre. We confirmedthat the survival disadvantage of mice globally expressing hy-peractivable V-NFAT1 or AV-NFAT1 was not due to deleteri-ous effects of expression of the Cre transgene (1). All offspringof R26STOPflox-V- or AV-NFAT1/R26� � CMV-CreCre/Cre crosses areexpected to express Cre, but only 50% (R26V- or AV-NFAT1/R26�,CMV-Cre) would delete the STOPflox cassette and express thehyperactivable NFAT1. The remaining 50% (R26�/R26�, CMV-Cre) were not only viable and healthy but were present in thelitters at much higher frequencies than expected because ofselection against the mice bearing the expressible transgene (Fig.2A). Thus, the observed developmental defects are not a resultof Cre recombinase expression but only become manifest whena hyperactivable NFAT1 is expressed.Tightly linked expression of hyperactivable NFAT1 and EGFP expressionfrom the ROSA26 locus. To confirm that EGFP expression wastightly linked to expression of hyperactivable NFAT1 from theROSA26 locus, splenocytes from a 12-week-old mosaic AVmouse (R26AV-NFAT1/R26�, CMV-Cre), which exhibited EGFPexpression in 16.2% of cells, were subjected to fluorescence-activated cell sorting (FACS) to separate EGFP-positive andEGFP-negative populations (Fig. S7A). These sorted popula-tions were then analyzed for transgenic NFAT1 expression byWestern blotting for the HA epitope tag. EGFP-negative spleno-cytes exhibited no expression, whereas EGFP-positive spleno-cytes showed clear expression of transgenic AV-NFAT1 (Fig.S7B), documenting that expression of hyperactivable NFAT1correlates unambiguously with EGFP expression in this exper-imental system.

SI Materials and Methods. Expression plasmids. The mutations FSILFto ASILA (amino acids 29–33, A) and SPRIEITPS toHPVIVITGP (amino acids 109–117, V) were introduced intopENTR11-NFAT1 (containing a cDNA of murine wild-typeNFAT1 with an N-terminal hemagglutinin epitope tag) by usingthe QuikChange II Site-Directed Mutagenesis Kit (Stratagene)to generate the constructs pENTR11-A-NFAT1, pENTR11-V-NFAT1, and pENTR11-AV-NFAT1. NFAT1 cDNA sequenceswere subsequently moved from pENTR11 to the retroviralexpression vector KMV by site-specific recombination usingGateway LR Clonase (Invitrogen). KMV is a modified Moloneyleukemia virus vector in which the attR site-f lanked cDNA isfollowed by a cDNA encoding EGFP under the control of anIRES.T cell isolation, retroviral transduction and differentiation. T cells wereisolated from mouse spleens and lymph nodes by using magneticbead selection. Cells were plated at a density of 1.52 � 106 CD8�

T cells per well in goat anti-hamster IgG-coated 12-well plates inthe presence of anti-CD3 (purified from supernatants of the2C11 hybridoma; 1 �g/mL) and anti-CD28 antibodies (1 �g/mL;37.51; BD PharMingen). For retroviral transduction, the cellswere spin-infected with supernatant from Phoenix packagingcells transfected with different KMV retroviral expression plas-mids as described previously (2). T cells were subsequentlyexpanded in vitro in the presence of IL-2 (20 U/mL).

Western blotting. For detection of NFAT1, monoclonal mouseanti-HA (1:1,000; 12CA5) or polyclonal rabbit anti-NFAT1(1:1,000; 67.1) was used. NF-�B p65 and Cre were detected byusing polyclonal rabbit anti-p65 (1:200; sc-372; Santa CruzBiotechnology) and polyclonal rabbit anti-Cre antibodies(1:1,000; ab 24608; Abcam).Flow cytometric analysis. For analysis of blood samples, a volume of�50 �L of peripheral blood was obtained from mouse tail veinsby using heparinized glass capillaries (Drummond). Blood cellswere washed twice in FACS buffer, stained for B220 [PE-conjugated anti-mouse CD45R (B220; eBioscience)], CD4(APC-conjugated anti-mouse CD4/L3T4; eBioscience), andCD8 (PerCP-Cy5.5-conjugated anti-mouse CD8a; BD Bio-sciences), and were subsequently analyzed by flow cytometry.Bone marrow, splenocytes, and thymocytes were stained by usingstandard protocols and by using the following antibodies: PerCP-Cy5.5-conjugated anti-mouse Sca-1, Alexa750-APC-conjugatedanti-mouse c-kit, Pacific Blue-conjugated anti-mouse lineage,Alexa647-conjugated anti-mouse CD34, PE-conjugated anti-mouse flk2, Cy7-PE-conjugated anti-mouse IL-7R� (all gener-ously provided by I. Weissman, Stanford University, Palo Alto,CA), Cy5-conjugated anti-mouse IgM (Jackson Laboratories),PE-conjugated anti-mouse CD25, PerCP-conjugated anti-mouseB220, PE-conjugated anti-mouse c-kit, PerCP-conjugated anti-mouse CD4, PerCP-conjugated anti-mouse CD8, APC-conjugated anti-mouse CD44, PE-conjugated anti-mouse TCR�(all from BD Biosciences), APC-conjugated anti-mouse CD4,and PE-conjugated anti-mouse B220 (eBioscience). Differentstages of B and T cell development were determined by using thefollowing surface marker profiles. Hematopoietic stem cells:Lin� Sca-1hi c-kithi (animal 1), Lin� Sca-1hi c-kithi f lk2� CD34�

(animals 2–5); common lymphoid progenitor: Lin� Sca-1lo c-kitlo

(animal 1), Lin� Sca-1lo c-kitlo f lk2hi IL-7R�� (animals 2–5);pro-B cells: IgM� B220�c-kit�; pre-B cells: IgM� B220�CD25�;immature B cells: IgM�B220lo; mature B cells: IgM�B220hi;splenic B cells: B220�; double-negative thymocytes 1 (DN1):CD44�CD25�; DN2: CD44�CD25�; DN3: CD44lo CD25�;DN4: CD44�CD25�; early double-positive thymocytes (DPs):CD4�CD8�TCR��; late DPs: CD4�CD8�TCR��; and single-positive thymocytes: CD4� or CD8�.NFAT1 nuclear translocation assay. CD8� T cells were collected onday 5 and plated at a density of 1 � 105 cells per well in a volumeof 200 �L of medium. The cells were subsequently stimulated forvarious time intervals with 10 nM PMA and 1 �M ionomycin.Calcineurin inhibition was performed with 1 �M cyclosporine(CsA). After stimulation, cells were attached to poly-L-lysine-coated wells in a 384-well plate by centrifugation for 3 min at149 � g (1.5 � 104 cells per well; 3 wells per sample), fixed with3% paraformaldehyde, and stained with either anti-NFAT1(purified rabbit polyclonal antibody to the 67.1� peptide ofNFAT1) or mouse anti-HA antibody. Indocarbocyanine-conjugated anti-rabbit or anti-mouse antibody was used assecondary antibody, and counterstaining of the nucleus wasperformed by using the DNA-intercalating dye DAPI. Imageswere acquired with the ImageXpress Micro automated imagingsystem (Molecular Devices) with a 20� objective and wereanalyzed with the translocation application module of MetaX-press 6.1 (Molecular Devices).

1. Schmidt-Supprian M, Rajewsky K (2007) Vagaries of conditional gene targetting. NatImmunol 8:665–668.

2. Macian F, et al. (2002) Transcriptional mechanisms underlying lymphocyte tolerance.Cell 109:719–731.

Muller et al. www.pnas.org/cgi/content/short/0813296106 1 of 10

Fig. S1. Effects of hyperactivable NFAT1 mutants on cytokine production. CD8 T cells from NFAT1�/� mice were retrovirally transduced to express wt orhyperactivable NFAT1, then left unstimulated or stimulated with PMA and increasing concentrations of ionomycin for 4 h. Production of TNF and IFN-� wascompletely inhibited by 1 �M CsA.

Muller et al. www.pnas.org/cgi/content/short/0813296106 2 of 10

Fig. S2. Generation of transgenic mice expressing hyperactivable NFAT1 from the R26 locus. (A) Targetting strategy. SA indicates splice acceptor site; pA,polyadenylation signal; DT, diphteroa toxin. (B) Southern blot of genomic DNA from targetted ES cell clones with a 5�-external probe (DNA digested with EcoRI;Left) or with an internal probe (DNA digested with BglI; Right).

Muller et al. www.pnas.org/cgi/content/short/0813296106 3 of 10

Fig. S3. Transient expression of Cre during early embryonic development in CMV-Cre transgenic mice. (A) Schematic illustrating X chromosome inactivationduring early embryonic development (1). Sperm containing an inactive X from the father (XP) fertilizes an oocyte containing an active X from the mother (Xm)to form a female zygote with 2 X chromosomes (2). After partial reactivation of XP in the zygote, another round of inactivation of the paternal X chromosome(XP) ensues, which then persists throughout the preimplantation period (3). After implantation of the blastocyst, the inner cell mass undergoes reactivation ofXP (4). Cells of the embryo proper then undergo random X chromosome inactivation, in which either Xm or XP are inactivated. Activities of the 2 X chromosomesduring these early stages of embryonic development are shown. (B) Cre expression in whole-embryonic lysates of CMV-Cre transgenic mice at embryonic day12.5. Lysates of CD4 T cells from B6 WT animals and CD4-Cre animals were used as negative and positive controls, respectively. Loading controls for NF-�B p65are shown. (C) Cre expression in peripheral T cells from CD4-Cre and CMV-Cre transgenic mice.

Muller et al. www.pnas.org/cgi/content/short/0813296106 4 of 10

Fig. S4. Deleterious effects of hyperactivable NFAT1 during early embryonic development. (A) Summary of crosses of heterozygous R26 transgenic mice(R26STOPflox-V- or AV-NFAT1/R26�) with homozygous CMV-Cre mice (CMV-CreCre/Cre) to achieve transgene expression early during embryonic development. (B)Male R26STOPflox-AV-NFAT1/R26STOPflox-AV-NFAT1 mice were crossed with female CMV-CreCre/� mice. Pregnant females were killed on E18.5 and embryos were assessedfor viability and genotype. Photograph of 2 independent litters at E18.5 are shown.

Muller et al. www.pnas.org/cgi/content/short/0813296106 5 of 10

Fig. S5. Germ-line selection against hyperactivable mutants of NFAT1. (A) Summary of crosses of mosaic R26 transgenic mice with C57BL/6 WT mice. (B) Expectedand actually detected frequencies of the recombined ROSA26 transgenes in offspring of different crosses. (C) Schematic overview of the efficiency of germ-linetransmission of the YFP, VIVIT-NFAT1, and AV-NFAT1 transgenes.

Muller et al. www.pnas.org/cgi/content/short/0813296106 6 of 10

Fig. S6. Flow cytometric analysis of peripheral blood from different ROSA26 transgenic mice. Peripheral blood from R26YFP/R26�, CMV-CreCre/� (n � 19);R26V-NFAT1/R26�, CMV-CreCre/� (n � 19); and R26AV-NFAT1/R26�, CMV-CreCre/� (n � 21) transgenic mice was drawn from tail veins by using heparin-coated glasscapillaries. Cells were stained with CD4-APC, CD8-PerCP and B220-PE conjugated antibodies and subsequently analyzed by flow cytometry. Representativehistograms for each group are shown.

Muller et al. www.pnas.org/cgi/content/short/0813296106 7 of 10

Fig. S7. FACS sorting of splenocytes from a mosaic AV mouse (R26AV-NFAT1/R26�, CMV-CreCre/�). (A) A single cell suspension was prepared from the spleen ofa 12-week-old R26AV-NFAT1/R26�, CMV-CreCre/� mouse. After osmotic lysis of erythrocytes, splenocytes were subjected to FACS sorting in EGFP-negative andEGFP-positive cell populations. Histograms of the unsorted and sorted populations are shown. (B) Western blot of EGFP-negative and EGFP-positive splenocytesfrom R26AV-NFAT1/R26�, CMV-CreCre/� mouse. Splenocytes were sorted, lysed, and subjected to SDS/PAGE and Western blotting. Mouse anti-HA antibody was usedto detect expression of the AV-NFAT1 transgene. Loading controls for NF�B p65 are shown.

Muller et al. www.pnas.org/cgi/content/short/0813296106 8 of 10

Fig. S8. Expression levels of hyperactivable NFAT1 in CD4 T cells. CD4 T cells were isolated from mice with the indicated genotypes and expanded undernon-skewing conditions. On day 6, cells were lysed and subjected to SDS/PAGE and Western blotting. Rabbit anti-NFAT1 antibody was used to detect expressionof endogeneous or transgenic NFAT1. Loading controls for NF�B p65 are shown.

Muller et al. www.pnas.org/cgi/content/short/0813296106 9 of 10

Fig. S9. Model for deleterious effects of hyperactivable NFAT1 early in embryonic development. Progenitor cells that escape the early wave of Cre-mediateddeletion and so lack expression of hyperactivable NFAT1 have a competitive advantage over cells expressing the hyperactivable protein. The extent of thisadvantage correlates with the degree of hyperactivability of the NFAT1 protein, but varies depending on the tissues to which the progenitor cells give rise. Theinner cell mass and the trophoblast are not distinguished for simplicity.

Muller et al. www.pnas.org/cgi/content/short/0813296106 10 of 10