siRNA-mediated reduction in MOLM-13-expressed mutant c-CBL transcript and protein is associated with reduced cell proliferation. (A) MOLM-13 cells were transfected (Nucleofector; Amaxa, Gaithersburg, MD) with siRNAs targeting the MOLM-13 c-CBL mutant transcript fusing exon 7 and exon 9 (siMolD8CBL) or a scrambled sequence (scrMolD8CBL) or transfected with “control siRNA” that has been validated to not affect mRNA levels in mammalian cells (Ambion, Austin, TX) and incubated at 37°C for 24 hours. Relative real-time RT-PCR was carried out using total RNA and mutant-specific primers and primers common to mutant and wild-type c-CBL transcripts (sequences available upon written request). 18S rRNA was used to normalize for starting template amounts. Expression of c-CBL WT or mutant transcript in each transfected sample was measured. Data are depicted as percent change in the mutant transcript level relative to the level of mutant in the control siRNA-transfected cells. No difference in c-CBL WT mRNA levels was observed (not shown). siRNA sequences are available upon request. (B) Immunoblot analysis of c-CBL protein in MOLM-13 cells transfected with no siRNA or either Scr or MolD8CBL siRNAs. Detection of actin was used to control for well loading. (C) MTS cell proliferation assay was carried out at 48 hours after transfection of MOLM-13 cells without (no siRNA) or with 1 nmol MolD8CBL or Scr siRNA. Viability is indicated as the average absorbance at 492 nm of triplicate wells corrected for background and relative to the 0-hour time point.