Figure 5: Strong positive correlation between Zeb2 and Il7r mRNA levels associates with increased JAK/STAT signalling.

(a) Quantitative Real-time (qRT)-PCR for Il7r in control P53/R26^+/+ (n=5) and Zeb2-overexpressing P53/R26-Zeb2^tg/+ (n=6) and P53/R26-Zeb2^tg/tg (n=6) thymic tumours. Mean±s.d. are indicated. (b) A correlation curve between Zeb2 and Il7r mRNA levels. Pearson Correlation coefficient (r) was calculated. (c) qRT–PCR for IL7R and ZEB2 in a cohort of human paediatric T-ALL patients; immature/ETP-ALL (n=9) and mature (n=8). A correlation curve between ZEB2 and IL7R mRNA is shown. Pearson Correlation coefficient (r) was calculated. (d) qRT–PCR for ZEB2 and IL7R after knockdown of ZEB2 using two independent siRNAs in the immature human T-ALL cell line LOUCY. Results are presented as averages±s.d. of three electroporations/siRNA. Student’s t-test was used for statistical analysis. (e) Genomic alignment with multiple species showing that a highly conserved region in the Il7r promoter is located 2.5–3.0 kbp upstream of the transcription initiation start site (red arrow). Localization of the amplicon used for ChIP is depicted. Figure was made using UCSC genome bioinformatics software (http://genome.ucsc.edu/). ChIP was performed using a specific anti-ZEB2 monoclonal antibody in a Zeb2-overexpressing T-ALL cell line. PCR amplification was analysed via agarose gel electrophoresis. Each lane is an independent immunoprecipitation. (f) Western blot analysis for total STAT5 and P-STAT5 levels in control P53/R26^+/+ and Zeb2-overexpressing P53/R26-Zeb2^tg/+ and P53/R26-Zeb2^tg/tg T-ALL cell lines with and without recombinant murine IL-7 administration. (g) Western blot analysis for total STAT5 and P-STAT5 levels in control and Zeb2-overexpressing mouse thymic tumours. (h) The pro-survival effects of IL-7 administration on cell survival of control and Zeb2-overexpressing mouse T-ALL cell lines cultured under stressed conditions. Every line represents the average of two-cell lines/genotype. Student’s t-test was used for statistical analysis. Knockdown experiments in d were repeated three times using triplicate biological samples. Western blots (f,g) were performed twice and representative blots and graphs are shown. Images have been cropped for presentation. Full size images are presented in Supplementary Fig. 12. Survival experiments (h) were performed twice. *P<0.05, **P<0.01, ***P<0.001 (vs control).