Fig. 5: Calcineurin blockade impairs new CD4 SP development and GATA3 induction.

a, Frequency (% of live cells) of CD4⁺CD8^lo, CD4 SM, CD4 SP or CD8 SP in thymic slices from postnatal (day 1) wild-type mice cultured with in 50, 100 or 200 ng ml⁻¹ cyclosporin A (CsA) for 96 h. Data were compiled from three independent experiments and analyzed using an ordinary one-way ANOVA. No CsA, n = 10; 50 ng ml⁻¹, 100 ng ml⁻¹ and 200 ng ml⁻¹, n = 7 each. Gating strategy, Extended Data Fig. 7a. b, Frequency (% of live cells) of CD4⁺CD8^lo and CD4 SM cells after 0, 24, 48, 72 and 96 h culture in medium alone or with 200 ng ml⁻¹ CsA as in a. Data were compiled from nine independent experiments. No CsA: 0 h, n = 6; 24 h, n = 9; 48 h, n = 10; 72 h, n = 22; 96 h, n = 10. 200 ng ml⁻¹ CsA: 24 h, n = 6; 48 h, n = 7; 72 h, n = 14; 96 h, n = 7. Data were analyzed using an ordinary two-way ANOVA with multiple comparisons. c, Frequency (% of live cells) of EdU⁺ DP, EdU⁺ CD4⁺CD8^lo, and EdU⁺ CD4 SP thymocyte populations from AND mice (i.p.) injected with EdU, rested for 16 h, and then treated with 1 (24 h) or 2 (48 h) 5 μg doses of FK506 (i.p.) every 24 h. Each dot represents one mouse. Data were pooled from three independent experiments. FK506 mice, n = 5; control, n = 4. d,e, Geometric mean fluorescent intensity (gMFI) of GATA3 detected by intracellular flow cytometry staining in CD69⁺ DP thymocytes from wild-type neonatal thymic slice cultures after 48-h treatment with 200 ng ml⁻¹ CsA (d) or 72-h treatment with 6.3 ng ml⁻¹ FK506 (e). Data were compiled from three (d) or two (e) independent experiments. For d, no CsA, n =  10 ; 200 ng ml⁻¹CsA, n = 10. Fore, no FK506, n = 6; 6.3 ng ml⁻¹ FK506, n = 6. Each symbol represents a thymic slice. Data were analyzed using an unpaired, two-sided t-test. norm., normalized; NS, not significant. Error bars indicate mean ± s.e.m.