NFAT5 Contributes to Osmolality-Induced MCP-1 Expression in Mesothelial Cells
Figure 3
NFAT5-knockdown attenuates osmolality-induced MCP-1 expression. Met5A cells were transfected with siRNA constructs for NFAT5 or with nontargeting siRNA as control as indicated. Cells were kept in isosmotic medium (gray column; 300 mosm/kg H2O) or were exposed to hyperosmotic medium (black column; 400 mosm/kg H2O). Medium osmolality was elevated by addition of glucose or NaCl as indicated, and cells were incubated for 24 h. (a) To demonstrate efficiency of NFAT5 knockdown, cells were processed for immunoblotting as described in Section 2. To demonstrate comparable protein loading, the blots were also probed for actin. (b) For determination of MCP-1 secretion, medium samples were collected and the concentration of MCP-1 in the cell culture supernatant was determined by ELISA as described in Section 2. Means ± SEM for per point; *. (c) For determination of MCP-1 transcription, RNA was extracted from the cells and the abundance of MCP-1 mRNA transcript was determined by qRT-PCR as described in Section 2. Relative MCP-1 mRNA abundance was normalized to that of β-actin to correct for differences in RNA input. Means ± SEM for per point; *.
