Fig 2: Pin1 ablation impairs Notch3 signaling in thymocytes of young N3IC 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), N3IC-tg (B) and N3IC-tg/Pin1−/− (C) mice. (b) Whole-cell extracts from thymocytes illustrated in (a) were revealed with anti-Pin1, anti-activated N3IC (N3IC-act), anti-HA (left panels) and anti-activated Notch1 (Notch1Val1744), 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), N3IC-tg (E) and N3IC-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 N3IC (N3IC-act), anti-HA and anti-β-actin antibodies and (e) in invasion Matrigel assay: relative percentage of DP cells invasiveness from N3IC-tg/Pin1−/− mice is shown with respect to N3IC-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.

Fig 3: Pin1 influences Notch3 processing and stability in endogenous and exogenous system. (a) Western blot analysis of Notch3 extracellular (N3EC) and activated intracellular (N3IC-act) protein expression of whole-cell extract (WCE) derived from Pin1-silenced TALL-1 (+) vs control cells (−) (left panel). The western blots in the figure are representative of at least three independent experiments, each in triplicate. The optical densitometry (OD) (right panels) was analyzed in all the experiments performed, thus including the P-values, calculated using Student's T-test (i.e., **P⩽0.01). (b) WCEs from Pin1-silenced TALL-1 cells (+) vs control cells (−) in a time course assay with 10 μg/ml of cycloheximide (CHX), in the presence or absence of the proteasome inhibitor MG132 for the same times before lysis, were revealed by immunoblotting with anti-activated N3IC (N3IC-act), anti-Pin1 and anti-β-actin antibodies (left panel). The right panel shows the relative quantification of activated-N3IC as determined by OD. (c) Left panel, Western blot analysis of whole-cell extracts from HEK293T cells transfected with Flag N3IC-wt plasmid and silenced for Pin1 (+) or control (−) in a time course assay with 10 μg/ml of cycloheximide (CHX). Extracts were immunoblotted with anti-Flag, anti-Pin1 and anti-β-actin antibodies. The right panel shows the relative quantification of Flag N3IC as determined by OD. All data are representative of at least three independent experiments, each in triplicate.

Fig 4: Notch3+CXCR4+DP T-cells increasingly infiltrate N3-ICtg BM with disease progression. In a, 3 weeks, 6–8 weeks, and 12–14 weeks of age WT (n = 4) and N3-ICtg (n = 6) BM. On the left of each figure section: representative plots of T-cell subset distribution. On the right: Notch3+CXCR4+ co-expression within DP-gated T-cells. The average percentages ± SD are shown. (**p<0.01; ns, not significant). b Absolute numbers of DP cells infiltrating transgenic (n = 3) BM (*p < 0.05, one-way ANOVA test). c Absolute numbers of Notch3+CXCR4+ and/or Notch3−CXCR4+DP-cells normalized to total DP gated cells of N3-ICtg bone marrow. For each corresponding age, at least five N3-ICtg mice were used. One-way ANOVA (Tukey’s post-test) and Student’s t-test performed where appropriate (**p < 0.01; ****p < 0.0001; ns, not significant)

Fig 5: In vivo CXCR4 antagonism greatly contrast atypical DP infiltration in N3-ICtg BM. Three- to four-week-old N3-ICtg mice were treated daily with PBS (PBS) or with the CXCR4 antagonist, AMD3100. At day10 post-injection, absolute numbers a of DP T-cells and b of Notch3+CXCR4+DP T-cells were analyzed. PBS, n = 3; AMD3100, n = 6. Results represent mean ± SD (*p < 0.05; **p < 0.01; Student’s t-test)