Fig. S3

Phenotypic analyses of foxp3a mutants. (A) Flow cytometry analysis of WKM from a representative Tg(foxp3a :EGFP); foxp3a(um252) animal. Gated lymphocytes are plotted as a histogram with the percentage of EGFP-positive lymphocytes indicated. (B) Percentages of foxp3a :EGFP -positive lymphocytes, based on flow cytometry analysis, are increased in a foxp3a(um252) mutant (n = 5) compared with a wild-type (n = 5) background. Two-tailed Student’s t test, *, P < 0.05. Error bar indicates SEM. (C) qRT-PCR of indicated genes in EGFP-positive lymphocytes sorted from Tg(foxp3a :EGFP); foxp3a(um252) and Tg(foxp3a :EGFP) animals. The log2 fold change of gene expression in EGFP-positive lymphocytes from Tg(foxp3a :EGFP); foxp3a(um252) animals compared with EGFP-positive lymphocytes from Tg(foxp3a :EGFP) animals is plotted. †, Expression of foxp3b was below the limit of detection in EGFP-positive lymphocytes from both Tg(foxp3a :EGFP); foxp3a(um252) and Tg(foxp3a :EGFP) animals. Error bar indicates SEM; n = 3. (D) Images of representative sibling foxp3a(um252)/+ heterozygotes and foxp3a(um252) homozygotes. (E) Normalized spleen sizes of sibling foxp3a(um252/+) (n = 4) and foxp3a(um252/um252) (n = 6) animals. Spleen sizes (μm² ×103/mm³) were estimated by normalizing maximum spleen area (area at level of splenic artery) by fish volume. Two-tailed Student’s t test, *, P = 0.007. (F) Flow cytometry analysis of a representative Tg(lck :EGFP) spleen. The lymphocyte-containing gate is indicated. (G) Gated lymphocytes from representative animals are plotted to indicate EGFP positivity. (H) qRT-PCR analysis of inflammation marker gene tnfa. ΔCt values were calculated relative to a β-actin control. Two-tailed Student’s t test, ns, not significant. Error bar indicates SEM; n = 3.