Fig. 6

Interaction of Nostrill and NF-кB p65 and impact of Nostrill induction on the transcriptional control of iNOS. a Physical interaction of Nostrill with NF-кB p65 in BV2 cells. BV2 cells were exposed to LPS (10 μg/ml) for 6 h, followed by RNA immunoprecipitation (RIP) analysis using anti-p65 or normal IgG. Presence of Nostrill, but not the control Actin in the immunoprecipitates from stimulated cells were detected by real-time PCR. ***p < 0.001 vs control IgG. †††p < 0.001 between indicated groups. b Diagram of primer sets of iNOS promoter region. c Impact of Nostrill on recruitment of NF-кB p65 to the iNOS gene locus in BV2 cells following LPS stimulation. BV2 cells were transfected with the Nostrill siRNA or control scrambled siRNA for 24 h, then stimulated with LPS (10 μg/ml) for 6 h, followed by ChIP analysis using anti-p65 and the PCR primer sets as designed. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control siRNA. †p < 0.05 and ††p < 0.01 between indicated groups. d Impact of Nostrill on recruitment of RNA polymerase II to the iNOS gene locus in BV2 cells following LPS stimulation. BV2 cells were transfected with the Nostrill siRNA or control scrambled siRNA for 24 h, then stimulated with LPS (10 μg/ml) for 6 h, followed by ChIP analysis using anti-Pol2 and the PCR primer sets as designed. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control siRNA. †p < 0.05 between indicated groups. e Impact of Nostrill on activating histone modifications at the iNOS gene locus in BV2 cells following LPS stimulation. BV2 cells were transfected with the Nostrill siRNA or control scrambled siRNA for 24 h, then stimulated with LPS (10 μg/ml) for 6 h, followed by ChIP analysis using anti-H3K4me3 and the PCR primer sets as designed. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control siRNA. †p < 0.05, ††p < 0.01, and †††p < 0.001 between indicated groups. f Increased recruitment of Nostrill to the iNOS gene locus in BV2 cells following LPS stimulation. BV2 cells were stimulated with LPS (10 μg/ml) for 6 h, followed by ChIRP analysis using two pools of probes specific to Nostrill and the PCR primer sets as designed. Probes for LacZ served as a negative control. *p < 0.05 vs even probe pool. #p < 0.05, ##p < 0.01 vs odd probe pool. Data represent means ± SEM from three independent experiments