Figure 2. Hyperosmolar stress-induced increase in MCP-1 mRNA and protein is independent of NFAT5 activation in ARPE-19 cells. ARPE-19 cells were transfected with sh-CTN plasmid (control) or sh-NFAT5 plasmid (A, B); with pEGFP (control) or DN-NFAT5 plasmid (C, D), then subjected to Iso (white columns) or Na100 (black columns) for 8 h, as previously described. MCP-1 mRNA levels measured by RT-qPCR (A, C) are expressed as MCP-1 mRNA levels (in fold expression) over Iso set to 1 following normalization with appropriate reference genes (YWHAZ, B2M for sh-NFAT5 [A]; YWHAZ, ATP5B for DN-NFAT5 [C]). MCP-1 protein levels quantified by ELISA are expressed as MCP-1 protein levels (in pg/ml; B, D). Data are the mean ± SEM of four (A, B) or three (C, D) independent experiments. Data were analyzed using repeated-measures analysis of variance with Bonferroni post hoc tests, the conformity t test, and the paired t test. *p < 0.05, **p < 0.01, ***p < 0.001.