Fig. 2

IL-20 expression was upregulated by NFAT5 activation under hyperosmotic stress. The hyperosmotic condition was achieved by adding various amounts of sodium chloride (NaCl) into the culture medium of HCE-2 cells. a, b Cells were treated with 0 mM, 20 mM, 40 mM, 60 mM, 80 mM, and 100 mM NaCl for 8 h. 40 mM NaCl was added into the media for 0, 2, 4, 6, 8, 12, and 24 h. The mRNA transcript of IL-20 was analyzed using real-time PCR with specific primers. GAPDH was used as an internal control. One-way ANOVA, **p < 0.01 and ***p < 0.001. Data are shown as the mean ± SEM. c, d HCE-2 cells were cultured without NaCl or with 40 mM NaCl + DMSO or 40 mM NaCl + KRN5 (an inhibitor of NFAT5) for 24 h. Total cell lysates and culture medium were harvested, and the IL-20 protein level was determined using an ELISA. One-way ANOVA, *p < 0.05 and **p < 0.01. Data are shown as the mean ± SEM. e Immunocytochemistry was also applied to analyze the protein levels of IL-20 and phospho-NFAT5 (Ser145). The reaction was detected using the chromogen AEC (red), and the nuclei were counterstained with hematoxylin (blue). Original magnification: ×200. f HCE-2 cells received control or hyperosmotic stress treatments (40 mM or 80 mM NaCl) to mimic dry eye conditions. mIgG (4 µg/ml) or 7E (4 µg/ml) were added to the medium for 8 h. The mRNA transcripts of TNF-α, MCP-1, ICAM-1, and MMP-9 were analyzed using real-time PCR with specific primers. GAPDH was used as an internal control. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Data are shown as the mean ± SEM. The experiments in a–f were independently repeated three times with similar results, and the data of one representative experiment is shown. NFAT5 nuclear factor of activated T‑cells 5, ELISA enzyme-linked immunosorbent assay