Figure 2
Activation of ISRE transcription by TNF-α does not require interferon production and the JAK-STAT signaling.
(A) qRT-PCR analysis of IRF1 overexpression by lentiviral vectors in the Huh7 based ISRE luciferase reporter cells. Compared to the control vector transduced cells, the IRF1 lentiviral vector showed strong IRF1 induction on RNA level. (B) Western blot analysis confirmed the successful overexpression of IRF1 by lentiviral vectors in the Huh7 based ISRE luciferase reporter cells. (C) In the Huh7 cell-based ISRE luciferase reporter cells, the combination of IRF1 over-expression and TNF-α induced a strong additive ISRE activation as measured at 3 different time points (n = 5). (D) qRT-PCR analysis of IRF1 knockdown by lentiviral shRNA vectors in the Huh7 based ISRE luciferase reporter cells. Compared to the control vector transduced cells, the IRF1 shRNA treated clones showed strong reduction of IRF1 RNA levels. (E) Western blot analysis confirmed the successful knockdown of IRF1 by lentiviral shRNA vectors in the Huh7 based ISRE luciferase reporter cells. (F) Knockdown of IRF1 in Huh7 based ISRE luciferase reporter cells did not block TNF-α induced ISRE-related luciferase activation (n = 4). (G) The relative IFN-α and β 1 expression levels in Huh7 cells were determined by qRT-PCR. GAPDH and RP2 served as internal reference genes. (H) IFN-α and β 1 expression levels in Huh7 cells were not up-regulated upon TNF-α treatment as measured by qRT-PCR (n = 6). (I) JAK inhibitor I (5 μ m) did not abrogate TNF-α induced ISRE-related luciferase activation (n = 3 independent experiments with 2–3 replicates each). Data presented as mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant).
