p66Shc deficiency enhances the ability of leukemic Eμ-TCL1 cells to promote homing chemokine expression in organotypic spleen cultures. (A) qRT-PCR analysis of the levels of CXCL9, CXCL10, CXCL11, CCL2, CXCL13, and CCL19 mRNA in spleen slices from WT mice kept in culture medium (n ≥ 6) or cocultured with conditioned supernatants (SN) from WT B cells (WT; n = 6) or p66Shc^−/− B cells (p66^−/−; n = 3) or leukemic B cells purified from spleens of Eμ-TCL1 mice (TCL1; n = 6) or Eμ-TCL1/p66Shc^−/− mice (TCL1/p66^−/−; n ≥ 6) with overt disease. The relative gene transcript abundance was determined on triplicate samples using the dCt method and normalized to GAPDH. Immunofluorescence (B) and flow cytometric (C) analysis of the percentage of CCL2⁺/podoplanin⁺ and CCL2⁺/ER-TR7⁺ cells in noncontiguous spleen slices from WT mice (n = 5) kept in culture medium (n ≥ 6) or cocultured with conditioned supernatants (SN) from WT B cells (WT; n = 6), or leukemic B cells purified from spleens of Eμ-TCL1 mice (TCL1; n ≥ 6) or Eμ-TCL1/p66Shc^−/− mice (TCL1/p66^−/−; n ≥ 6) with overt disease, and stained with anti-podoplanin and anti-CCL2 antibodies or with anti–ER-TR7 and anti-CCL2 antibodies. Immunofluorescence images were acquired on a confocal microscope using a 10× objective. Representative immunofluorescence images and flow cytometric plots are shown. Scale bars, 100 μm. Bar graphs in (A) and (B) show mean ± standard deviation. ***P ≤ .001, **P ≤ .01, *P ≤ .05, Student t test. co-loc, co-localization.