Figure 8
Indirubin derivatives suppress H9N2-induced IFN-β mRNA expression through inhibiting STAT3 signaling pathway.
(A,B) HPMECs transfected with non-targeting siRNA (50 nM) or STAT3 specific siRNA (50 nM) were infected with H9N2 at 2 MOI for 1 h and further incubated for 24 h.p.i. The mRNA levels of IFN-β and IFN-γ1 were quantified by real-time RT-PCR. (C) HPMECs were infected with H9N2 at 2 MOI followed by treatment with indirubin derivatives E804 or E231 (1 μM). Total cell lysates were harvested at 2 h.p.i. Phosphorylation and total STAT3 were detected by Western blot analysis with specific antibodies. (D) Subcellular fractionation was performed 2 h post infection. Western blot analysis was performed to determine the translocation of STAT3 from cytoplasm to nucleus. The GAPDH and Lamin A/C denoting the cytoplasmic and nuclear fractions respectively. Representative images from three independent experiments (Upper panel). Band intensities were determined by quantitative densitometry. Values are presented as fold-change of phosphorylated STAT3 normalized to total STAT3 and then compared with mock-infected cells (Lower panel). The values are presented as mean ± S.D. from three independent experiments. ***p < 0.001 vs mock-infected cells, ###p < 0.001 vs H9N2-infected cells. (E) Immunofluorescent staining of HPMECs with anti-phosphorylated STAT3 (Y705) antibody (Green). DAPI staining (Blue) was used to show the nucleus.
