Role of Klf4 in iNOS expression upon LPS stimulation. (A) RT-PCR for iNOS mRNA demonstrates a significant decrease in iNOS mRNA levels upon Klf4 knockdown compared to LPS-treated cells. (B) Immunoblot showing iNOS levels under different conditions. There is a significant decrease in iNOS proteins levels in Si+LPS cells compared to LPS-treated cells. The graphs represent relative iNOS mRNA and protein levels with respect to the untreated controls. (C) Nitrite assay using Griess reagent was carried out to measure iNOS activity. A significant reduction is noticed in NO production as a result of Klf4 knockdown in LPS-treated samples. (D) Luciferase assay for iNOS promoter activity. Data is represented as relative luciferase units/amount of protein (in μg). A more-than-3-fold decrease is observed in luciferase activity in Si+LPS cells compared to LPS-treated cells. (E) EMSA carried out with nuclear extracts of control and LPS-treated BV-2 cells. Lane 1 shows free iNOS probe, whereas a shift is noticed in lane 2 when nuclear extracts were incubated with the probe. Lane 3 shows a supershift when Klf4-specific antibody was incubated with the probe and the nuclear extracts. The shift and supershift are indicated by arrows. Lane 4 shows a decreased shift when nuclear extracts from unstimulated control BV-2 cells were incubated with the iNOS probe. In lane 5, in addition to biotinylated probe, a 100-molar excess of unbiotinylated (Cold) probe was added along with nuclear extracts from LPS-stimulated cells. No significant band is observed in this lane. *, **, Statistical differences in comparison to control values and #, Statistical differences with respect to LPS-treated values respectively (* p < 0.05, ** p < 0.01, # p < 0.01).