Figure 2. Lin28 positively affects the expression of BMP4 at the protein level. IGROV1 (A), A2780 (B) or PA-1 (C) cells were transfected with empty vector, FL-Lin28, siCon or siLin28 as indicated. Proteins and RNAs were extracted 48 h post-transfection and levels determined by western blotting and RT-qPCR analysis, respectively. Representative results of two independent experiments for each cell line are shown. For protein analysis, β-tubulin was used as a loading control. Protein bands on western gels were quantitated using the Bio-Rad Quantity One software. For RNA analysis, β-tubulin mRNA was used for RNA level normalization, and β-actin mRNA was used for non-target control for Lin28 effects. The levels of BMP4, β-actin and Lin28 mRNAs from empty vector- or siCon-transfected cells were arbitrarily set as 1. Error bars are mean ± SD (n = 3). (D) Reporter assays. Top: a schematic drawing of human BMP4 transcript. The red rectangle represents open reading frame, with the 411-nt LRE highlighted in blue. Numbers are in nucleotides relative to the transcriptional start site of BMP4. Outlined beneath is a firefly luciferase reporter construct (FFL) with the green box representing its coding region and the thin line representing 3′-UTR. The position where the LRE was inserted is indicated. Bottom: luciferase assay results. The indicated constructs were each transfected into HEK293 cells that do not express endogenous Lin28, with increasing amounts of co-transfected FL-Lin28. Luciferase activities and FFL mRNA levels were measured 24 h post-transfection. Relative luciferase activities were presented after normalization against FFL mRNA levels. Luciferase activities from cells without FL-Lin28 transfected were arbitrarily set as 1. Numbers are mean ± SD (n = 3).