Manipulations of MYB and miR-155 levels in B-CLL. (A) Transfection of primary B-CLL cells (P88) and Raji cell line with siRNA oligos inhibiting MYB expression (final concentrations of 30nM, gray bars; and 100nM, black bars) or with negative control oligos. The y-axis indicates quantitative PCR-determined relative levels of mRNA of indicated genes related to housekeeping gene GAPDH (for MYB, PU.1) or to RNU44 (for miR-155) 48 hours after transfection (“Transfections”). Data (shown as a fold change [FC]) are normalized to mRNA levels obtained in negative control transfection experiment (control measurements were set as baseline = 1). Error bars represent SEM. *P < .05. **P < .01. (B) Raji cells were transfected with MYB cDNA encoding expression plasmid (in 2 final concentrations: 0.2 μg/mL, gray bars; and 2.0 μg/mL, black bars) or with a negative control (pcDNA 3.1) plasmid. The y-axis: quantitative PCR-determined relative levels of mRNA miR-155 compared with the RNU44 internal control (control measurements were set as baseline = 1) 48 hours after transfection (“Transfections”). Error bars represent SEM. *P < .05. **P < .01. Immunoblotting of MYB (and β-actin) in HeLa cells at 48 hours after transfection indicates expression product of the MYB cDNA expressing plasmid. The relative optical density of each protein is indicated below the blots. (C) B-CLL cells (P161) and Raji cell line was transfected with anti–miR-155 oligos (at final concentration of 40nM, gray bars; and 100nM, black bars) and with negative control oligos. After 96 hours, the total RNA was purified, reverse transcribed, and measured by quantitative PCR as described in “RNA expression.” The y-axis indicates relative expression of mRNA of indicated genes relative to the housekeeping gene GAPDH (for PU.1) or to RNU44 (for miR-155). Data are normalized to the mRNA levels obtained in negative control transfection experiment (control measurements were set as baseline = 1). Error bars represent SEM. *P < .05. **P < .01. (D) Raji cell line was transfected with miR-155 oligos (at final concentration 40nM, gray bars; and 100nM, black bars) or with negative control oligos. After 24 hours, total RNA was purified, reverse transcribed, and measured by quantitative PCR as described in “RNA expression.” The y-axis indicates relative expression of mRNA of indicated genes relative to the housekeeping gene GAPDH (for PU.1) or to RNU44 (for miR-155). Data are normalized to the mRNA levels obtained in negative control transfection experiment (control measurements were set as baseline = 1). Error bars represent SEM. *P < .05. **P < .01.