Figure 1.

Hypertonicity induces TNF transcription and NFAT5 expression. (A) Induction of TNF mRNA by hypertonic stress. L929 cells were cultured in medium supplemented with 200 mM raffinose for 18 h. RNA was harvested and submitted to RNase protection assay. (B) Hypertonic stress up-regulates NFAT5 expression. L929 cells were cultured to ∼95% confluence in a 6-well plate and stimulated with complete medium or medium supplemented with 200 mM raffinose for 18 h, as indicated. Whole cell extracts were collected and submitted to western blot analysis. Results are representative of four independent experiments. (C) L929 cells do not express NFATp. Whole cell extract (30 μg) from 68 to 41 cells were submitted to western blot analysis as a positive control for NFATp expression. L929 cells were stimulated with hypertonic stress as above. Results are representative of two independent experiments. Lamin B1 was used as a loading control. (D) Induction of TNF luciferase activity by hypertonic stress in L929 fibroblasts. A total of 2 × 10⁶ L929 cells were transfected with 1 μg −200 TNF-Luc or −982 TNF-Luc and 0.4 μg pTK-RL control vectors by DEAE-dextran. Cells were stimulated with complete medium supplemented with 200 mM raffinose 24 h after transfection. Cells were harvested 18 h after stimulation and luciferase activities were assayed. Results are from four independent experiments. Error bars are the standard deviation.