CD44 cleavage is required for IFN-γ-induced expression of IFI16.

(A) RPM-MC cells were transfected either with full-length CD44WT or CD44-KR-MT, serum starved O/N and subsequently treated with 100ng/ml TPA for 30 min. After additional 12 or 6 hrs of cultivation in serum-free medium, total RNA was isolated and used for RT-qPCR. Similar results were obtained in 3 separate experiments (IFI16: interferon-inducible protein 16: IFItm3: interferon-inducible transmembrane protein 3). (B-E) RPM-MC cells were seeded in 6 cm dishes at a cell density of 3x105 cells/dish. 24 h after seeding, cells were transiently transfected with either a control vector or with vectors expressing either CD44ΔE (mimicking the product of ADAM cleavage, which serves as substrate for γ-secretase), CD44-ICD or CD44 full length. For cell recovery, medium was changed after 14 h. Subsequently, cells were stimulated with 100 ng/ml IFN-γ for 6 h. RNA was isolated and transcribed into cDNA. cDNA was analysed by RT-qPCR using primers for human IFI16 (primers 9 and 10, Table 1) and actin for normalization (primers 7 and 8, Table 1). In Fig 2C γ-Secretase activity was blocked by 5 μM DAPT O/N prior to IFN-γ treatment. All experiments were done in triplicates. For statistical analysis see Materials and methods.