Hypertonicity-induced NF-κB activation is dependent on IKKβ
activity, IκBα degradation, and p65 activation. A and
B, cells were transfected with an expression vector that contained
either eGFP or constitutively active IKKβ or IκBα mutants
together with (A) or without (B) NF-κB-driven
luciferase plasmid. Cells were challenged or not with either 100 μg/ml LPS
or hypertonic (NaCl, 500 mosmol/kg) medium for 12 (A) or 3
(B) h before measurement of luciferase activity (A) or to
RNA extraction (B). Real-time PCR was performed using primers
specific for TNFα (B). Results are expressed relative to
control values determined in cells transfected with eGFP-containing cDNA and
subjected to isotonic medium. Bars are the mean ± S.E. from
four independent experiments. *, p < 0.05. C, cells were
transfected with either scrambled RNAi or RNAi targeting p65 NF-κB and
then challenged or not with either 100 ng/ml LPS or hypertonic (NaCl, 500
mosmol/kg) medium for 3 h before RNA extraction. Real-time PCR was performed
using primers specific for MCP-1, IκBα, or TNFα. Results are
expressed relative to control values determined in cells transfected with
scrambled RNAi and subjected to isotonic medium. Bars are the mean
± S.E. from four independent experiments. *, p < 0.05.
Ctl, control.