Figure 5

Enhanced NF-κB activation in TNF-α- or IL-1β-primed HSCs after stimulation with peptidoglycan or lipoteichoic acid. (a) Activated human HSCs were infected with Ad5NF-κBLuc (MOI 500) for 12 h in DMEM containing 0.5% FCS. HSCs were then incubated in serum-free DMEM with TNF-α (10 ng/ml) or IL-1β (10 ng/ml) for 24 h. After washing three times, HSCs were stimulated with media, PGN (S. aureus) (10 μg/ml), PGN (B. subtilis) (10 μg/ml), or LTA (S. pyogenes) (10 μg/ml) for 8 h in serum-free conditions. Data represent the mean±s.d. of three independent experiments performed in duplicate, and are expressed as fold-increase over unstimulated cells. All measurements of luciferase activity were normalized to the protein concentration. ^*, †, ‡: P<0.01, when compared to control HSCs without stimulation; §: P<0.01, unprimed HSCs vs TNF-α-primed HSCs; ^**: P<0.01, unprimed HSCs vs IL-1β-primed HSCs; #: P<0.01, TNF-α-primed HSCs vs IL-1β-primed HSCs. (b) Activated human HSCs were incubated with DMEM alone (lanes 1–3) or with TNF-α (10 ng/ml) for 24 h (lanes 4–8). Cells were then treated as follows: lanes 1,4, DMEM; lanes 2,5, PGN from S. aureus (10 μg/ml); lanes 3,6, LTA from S. pyogenes (10 μg/ml); and lane 7, IL-1β (5 ng/ml). Lanes 8 is the same as lane 5 with the addition of a 100-fold molar excess of cold oligonucleotide as a competitor. Nuclear extracts (5 μg) were assayed for NF-κB binding activity by EMSA using a radiolabeled consensus NF-κB site as a probe. A representation of three independent experiments is shown. ^*, †: P<0.01, when compared to control HSCs without stimulation; ‡: P<0.01, unprimed HSCs vs TNF-α-primed HSCs.