Fig 3. IFI16 up-regulation is implemented at the level of transcription.

(A) RPM-MC cells were prepared as in Fig 2 and transfected with a control vector or a vector expressing the CD44-ICD as described above. (B) Immediately before treating cells with IFN-γ (100 ng/ml, 6 h), actinomycin D (5 μg/ml) was added to the cells. RNA was analysed as described in Fig 2. (C) RPM-MC cells were seeded in 24-well plates at a density of 4x10⁴ cells/well. 24 h later cells were transiently transfected with either a control vector or CD44-ICD together with a construct of collagenase 1 promoter fused to the firefly-luciferase gene. Transfection medium was exchanged to fresh medium after 14 h. Overnight treatment with IFN-γ (100 ng/ml) was followed by analysis. Luminescence was measured after addition of Firefly-luciferase substrate. (D) Cells were transiently transfected with GFP-tagged wild type or KR-MT intracellular domain of CD44 (CD44-ICD). After 24 h, cells were fixed with 4% PFA and examined by microscopy (Axiovert 135). RPM-MC cells: 46 cells of total 50 cells showed nuclear localisation of wild type CD44-ICD; 32 cells of total 50 cells showed cytoplasmic localization of mutant CD44-ICD. RT4-D6P2T cells: 45 cells of total 50 cells showed nuclear localisation of wild type CD44-ICD; 30 cells of total 50 cells showed cytoplasmic localization of mutant CD44-ICD. Experiments were done in triplicates. For statistics see Materials and methods.