Figure 3

Ik-1 and MZF1 decrease IGF-IR mRNA and protein levels and the phosphorylation of downstream targets. (A) Western blotting demonstrates increased expression levels of Ik-1 and MZF1 proteins at 48 h after transfection into 3 NPM-ALK⁺ T-cell lymphoma cell lines. β-actin shows equal protein loading (−: transfection of EV; +: transfection of Ik-1 or MZF1). (B) Transfection of Ik-1 remarkably decreased IGF-IR mRNA levels in Karpas 299, SUP-M2, and SR-786 cell lines (*: < 0.0001 compared with EV). (C) Similarly, transfection of MZF1 induced a significant decrease in IGF-IR mRNA levels (*: < 0.0001 compared with EV). The results depicted in (B) and (C) represent the means ± SE of 3 experiments. (D) Western blotting shows that transfection of Ik-1 and MZF1 into Karpas 299, SUP-M2, and SR-786 cell lines induced marked downregulation of IGF-IR protein, which was associated with decreased pIGF-IR levels. Moreover, the decrease in IGF-IR/pIGF-IR expression levels was associated with decreased phosphorylation of important IGF-IR targets including IRS-1, AKT, and NPM-ALK. Whereas basal levels of AKT remained unchanged, notable decrease in IRS-1 protein was observed. The 3 web-based transcription factor search algorithms showed that Ik-1 and MZF1 could potentially bind the IRS-1 gene promoter. In contrast, searching these algorithms did not support potential binding of Ik-1 or MZF1 and the NPM gene promoter, where the expression of NPM-ALK protein is regulated at the transcriptional level. (E) In line with lack of potential binding/interaction between Ik-1 or MZF1 and the NPM gene promoter, a luciferase assay performed in R⁻ cells shows that transfection of Ik-1 and MZF1 does not decrease the NPM promoter activity. The results represent means ± SE of 3 consistent experiments.