p66Shc reconstitution in CLL B cells results in enhanced S1P1 expression. qRT-PCR (A) and flow cytometric (B) analysis of S1P1 expression in PB B cells purified from CLL patients with either mutated (M; n = 7) or unmutated (U; n = 9) IGHV nucleofected with either empty vector (vector ctr) or an expression construct encoding p66Shc (p66). The analysis was carried out 48 hours after transfection on GFP⁺ live cells. All samples were checked for reconstitution of p66Shc expression by qRT-PCR (data not shown). An immunoblot analysis of p66Shc and S1P1 expression on a representative experiment is shown on the right. The relative abundance of gene transcripts was determined on triplicate samples from each patient using the ddCt method and is expressed as the normalized fold expression (mean ± SD; empty vector controls taken as 1 for all CLL samples). The data in the histograms showing the quantification of surface S1P1 are expressed as the mean fluorescence intensity of p66-transfected cells or of vector ctr-transfected cells (normalized to 1 in all empty vector controls) ± SD. ***P < .001; **P < .01; and *P < .05.