p66Shc deficiency in leukemic Eμ-TCL1 cells results in enhanced IL-9 expression. (A) Heat map and hierarchical clustering of the Immune System process transcripts from Affymetrix array analysis showing differential expression patterns between leukemic Eμ-TCL1 cells (TCL1; n = 3) and Eμ-TCL1/p66Shc^−/− (TCL1/p66^−/−; n = 3) cells. Differential expression criteria: P value < .05, estimated fold change > 2. Upregulated and downregulated transcripts are shown in red and blue, respectively. (B) Fold change and P value of il-9 expression extrapolated from Affymetrix array analysis in (A). (C) qRT-PCR analysis of IL-9 mRNA in WT B cells (n = 11), p66Shc^−/− B cells (n = 9), or leukemic B cells purified from Eμ-TCL1 mice (n = 12) or Eμ-TCL1/p66Shc^−/− mice (n = 18) mice with overt disease. (D) Quantification by ELISA of IL-9 released in the culture supernatants of WT B cells (n = 6), p66Shc^−/− B cells (n = 9), or leukemic B cells purified from Eμ-TCL1 mice (n = 10) or Eμ-TCL1/p66Shc^−/− mice (n = 23) with overt disease. qRT-PCR analysis of CCL2 mRNA (E) and quantification by ELISA of CCL2 released in the culture supernatants (F) from OP9 stromal cells kept in culture medium or stimulated with 0.05 ng/mL IL-9 alone or in combination with 0.1 ng/mL isotype-control antibody (ctr Ab) or 0.1 ng/mL neutralizing anti–IL-9 mAb (IL-9 Ab) for 48 hours (n = 3). (G) qRT-PCR analysis of CCL2 mRNA in OP9 stromal cells kept in culture medium or cultured for 48 hours with conditioned supernatants from leukemic B cells purified from Eμ-TCL1 mice (n = 3) or Eμ-TCL1/p66Shc^−/− mice (n = 3) with overt disease and with 0.1 ng/mL isotype-control antibody (ctr Ab) or 0.1 ng/mL neutralizing anti–IL-9 antibody (IL-9 Ab). The relative gene transcript abundance was determined on triplicate samples using the dCt method and normalized to GAPDH. Data are mean ± standard deviation. ***P ≤ .001, **P ≤ .01, *P ≤ .05, 1-way ANOVA, multiple comparisons.