Figure 1

NFAT5 differentially affects nuclear factor-κB (NF-κB) activity in macrophages in a stimulus-dependent manner. (a and b) Additive effects of LPS and NaCl on NFAT5 activation. LPS (5 μg ml⁻¹) or heat-inactivated E. coli (3 × 10⁶ CFU ml⁻¹) were added to RAW 264.7 cells for 24 h in the presence or absence of NaCl (90 mM). NFAT5-green fluorescent protein (GFP) activity and NFAT5 translocation were determined by flow cytometry and western blot analysis, respectively. The data on the right in a show the mean±s.d. of three independent experiments. *P<0.01 versus LPS or heat-inactivated E. coli alone. (c) LPS- and high salt-induced increases in NFAT5 reporter activity in vivo. Matrigel with RAW 264.7 macrophages stably transfected with NFAT5-red fluorescent protein (RFP) reporter were subcutaneously implanted into mice. On day 9, LPS (10 mg kg⁻¹) and hypertonic saline (11.3% HS, 25 cc kg⁻¹) were injected intraperitoneally. Normal saline (0.9% NS, 25 cc kg⁻¹) was injected as a control. At 16 h post injection, NFAT5-RFP activity in the Matrigel was determined using the Maestro Imaging System. (d) Transcriptional activity of NF-κB in NFAT5-deficient macrophages treated with LPS (5 μg ml⁻¹) or NaCl (90 mM). An NF-κB reporter containing GFP was transiently transfected into cells, and GFP activity representing NF-κB was determined by flow cytometry (left). The bar graph on the right represents the mean±s.d. of five independent experiments. *P<0.05 versus control short hairpin RNA (shRNA)-transfected cells in the presence or absence of NaCl. The reduction in NFAT5 expression was confirmed by western blot analysis (top panel).