Figure 2

PCBP1 inhibits CD44 v5 splicing. (A) Schematic representation of the luciferase splice reporter construct pETCatEBLucv5. (B) The pETCatEBLucv5 vector was cotransfected in HepG2 cells with different doses of pcDNA3.1(His/Myc)-PCBP1 vector and a Rennila luciferase plasmid served as an internal transfection control. The expression of overexpressed PCBP1 and endogenous PCBP1 was investigated by Western blotting. The blots were scanned for densitometry analysis with the value obtained from control cells set as 1. The values were normalized with those of Actin. The overexpressed PCBP1 levels were quantified relative to the endogenous PCBP1 expression level. 24 hours later cell lysis were prepared for the luciferase activity assay (C) and total RNA were extracted for RT-PCR (D). The level of luciferase was normalized to 100 following transfection with the control plasmid. The inclusion and exclusion fragments were produced by the primers indicated in (A). Values for exon inclusion were indicated. (E) HepG2 cells were co-transfected with pETCatEBLucv5 vector along with either negative control siRNA (N.C) or PCBP1 specific siRNA oligos for 48 hours and then luciferase activity was measured and the inclusion and exclusion fragments were analyzed by RT-PCR (F). Statistical analysis was performed and the results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as ** p ≤ 0.001.