(A) RAW 264.7 MΦ (left panel) and bone-marrow-derived MΦ (BMM, right panel) were cultured in normal cell culture medium (NS: normal salt), with additional 40 mM NaCl in the medium (HS: high salt), or 80 mM urea ± 10 ng/ml LPS for 24 hr. Nos2 mRNA (mean + SEM; n = 4 [RAW264.7]; n = 4 to 5 [BMM]), ∗ p(HS) < 0.05; # p(LPS) < 0.05; † p(LPS∗HS) < 0.05; NOS2 protein, and nitrite levels (mean + SEM; n = 4 [RAW264.7]; n = 11 [BMM]); Triangles: not detectable (n.d.).

(B) BMM were cultured in NS, with HS ± LPS (1 ng/ml), IL-1α (50 ng/ml) or IL-1β (50 ng/ml) + TNF (20 ng/ml) for 24 hr. Nitrite levels (mean + SEM; 4 similar experiments); Triangles: n.d.

(C) RAW 264.7 MΦ were cultured in NS, with HS or 80 mM urea ± LPS (10 ng/ml) for 45 min. Upper panel, densitometry and immunoblotting of p38/MAPK and activated p-p38/MAPK (mean + SEM; n = 8). # p(LPS) < 0.05; † p(LPS∗HS) < 0.05. Lower panel, immunoblotting detected the p38/MAPK substrate MK2 and activated p-MK2.

(D) RAW 264.7 MΦ were pretreated ± p38 blocker SB203580. After 0.5 hr cells were cultured in NS, with HS ± 10 ng/ml LPS for 24 hr. Upper panel, TNF levels (mean + SEM; n = 2 in triplicates); lower panel, nitrite levels (n = 7). Triangles: n.d.

(E) BMM from Poly(I:C)-treated MxWT p38αfl/fl (control) and MxCre p38αfl/fl (Δ p38α) mice were cultured in NS, with HS ± LPS (1 ng/ml) for 24 hr. Upper panel, nitrite levels (mean + SEM; n = 2 in quadruplicates); Triangles: n.d.; Lower panel, immunoblotting of p38/MAPK and HSP90.

(F) As in (D). Immunoblotting of NFAT5 and actin.

(G) RAW 264.7 MΦ electroporated with control non-silencing siRNA or Nfat5-specific siRNA (Nfat5 siRNA) were cultured in NS or HS ± LPS (10 ng/ml) or LPS/IFN-γ under NS for 24 hr. Triangles, n.d. Immunoblotting of NFAT5 and Actin. Nitrite levels (mean + SEM; n = 3 to 4).

(H) RAW 264.7 wild-type MΦ (Nfat5 wt) and RAW 264.7 MΦ with stable Nfat5 overexpression (Nfat5-over) were cultured NS or HS ± LPS (10 ng/ml) for 24 hr. Immunoblotting of NFAT5 and actin. Nitrite levels (mean + SEM; n = 4).

(I) As in (D), but in addition RAW 264.7 MΦ with stable Nfat5 overexpression (Nfat5-over) were used. A representative experiment in quintuplicates out of two independent experiments is displayed (mean + SEM).

(K) Schematic of HS-induced alterations in MΦ LPS signaling.