Figure 6

(a) FTS displaces the transcription factor IRF7 from the nucleus. The cells were treated similarly and then stained for confocal microscopy using anti-IRF7 Ab (see Materials and methods section). The nuclear localization of IRF7 was quantified using Hoechst staining of the nucleus (white arrow heads). Typical images of the cells are shown. (b) The cells were treated similarly and then subjected to nuclear fractionation (see Materials and methods section). IRF7 levels are shown in the nucleus and the cytosol. PARP and β-tubulin were used as nuclear and cytosolic markers, respectively, in order to show the quality of fraction separation. (c) Histograms of the IRF7 nuclear localization ratio (nuclear fluorescence/total cell fluorescence) as shown in a. FTS or rapamycin treatment or TSC2 re-expression decreased the nuclear fraction of IRF7 (means±S.E.M., n=30, *P<0.05, **P<0.01, ***P<0.001). Similar results were obtained from three separate experiments. (d) Histograms of the IRF7 nuclear localization ratio (nuclear/cytosol intensity) as shown in b. FTS or rapamycin treatment or TSC2 re-expression decreased the nuclear fraction of IRF7 (means±S.E.M., n=3, *P<0.05, **P<0.01). (e) Knockdown of IRF7 mimics the treatment with FTS or rapamycin or re-expression of TSC2. The 621.102 cells were transfected for 72 h with non-targeting control or IRF7 siRNA. The mRNAs of the indicated genes were then quantified with specific primers by qRT-PCR (see Materials and methods section). Their quantified levels are shown (means±S.E.M., n=3, *P<0.05, **P<0.01, ***P<0.001). (f) After transfection, 621.102 and 621.103 cells were re-seeded for 72 h and then counted to evaluate their proliferation rates. The percentage of cells relative to the control siRNA is shown. Downregulation of IRF7 reduced the proliferation rate of the 621.102 cells but did not change the proliferation rate of the 621.103 cells (means±S.E.M., n=4, **P<0.01). (g) After transfection, 621.102 and 621.103 cells were re-seeded for 48 h and then fixed, PI stained and subjected to FACS for cell cycle analysis (see Materials and methods section). The quantification of the different cell cycle phases is shown. No significant difference was observed after IRF7 knockdown