Figure 3

Pin1 ablation impairs Notch3 signaling in thymocytes of young N3_IC transgenic mice resulting in the decrease of expansion/invasiveness of CD4⁺CD8⁺ DP splenic cells. CD4⁺ and/or CD8⁺ subset distribution of thymocytes from representative 6-week-old Pin1^+/+ (A), N3_IC-tg (B) and N3_IC-tg/Pin1^−/− (C) mice. (b) Whole-cell extracts from thymocytes illustrated in (a) were revealed with anti-Pin1, anti-activated N3_IC (N3_IC-act), anti-HA (left panels) and anti-activated Notch1 (Notch1_Val1744), anti-Hes1 and anti-pTα (right panels) antibodies. Western blot against the anti-β-actin was used as a loading control. (c) CD4⁺ and/or CD8⁺ subset distribution of lymphocytes derived from SPL and blood of representative 6-week-old Pin1^+/+ (D), N3_IC-tg (E) and N3_IC-tg/Pin1^−/− (F) mice. (d) Sorted CD4⁺CD8⁺ (DP) splenocytes illustrated in (c) (circle around the number) were used for western blot analysis against anti-activated N3_IC (N3_IC-act), anti-HA and anti-β-actin antibodies and (e) in invasion Matrigel assay: relative percentage of DP cells invasiveness from N3_IC-tg/Pin1^−/− mice is shown with respect to N3_IC-tg cells. Results are shown as the means average deviations of five independent experiments (n=3–5 mice per group) and P-values were calculated using Student’s T-test (i.e., **P⩽0.01). In all panels described in (a, c), numbers inside each cytogram indicate the percentages of the corresponding subsets and the results are representative of five independent experiments (n=3–5 mice per group: Pin1^+/+ (n=15), N3IC-tg (n=25) and N3IC-tg/Pin1^−/− mice (n=15)). THY, thymus. SPL, Spleen; PB, Peripheral Blood.