Fig. 5.

Fate of MRTF under strongly hyperosmotic conditions. A: following serum depletion, LLC-PK1 cells were incubated in serum-free isotonic or hypertonic DMEM. The osmolarity of the medium was set between 500 and 700 mosM (total) by the addition of extra NaCl at the indicated concentrations. After 24 h of exposure, the cells were lysed and whole cell lysates were subjected to immunoblotting for the indicated proteins. The graph below the blot shows densitometric quantification of MRTF protein levels normalized to the corresponding GAPDH levels (means ± SE, n = 3). B: cells were incubated for 2 h in serum-free isotonic or hypertonic DMEM supplemented with extra NaCl at the indicated concentrations and then lysed and processed as in A. Note the robust decrease in the MRTF signal at and above a total osmolarity of 700 mosM. C: short-term exposure to strong hyperosmolarity (≥700 mosM) activates caspase-3 in LLC-PK1 cells. Cells were treated for 2 h with the indicated extra concentrations of NaCl added into the isotonic Na⁺ medium. Cells were harvested and lysed, and the obtained lysates were assayed for caspase-3 activity, using a fluorigenic substrate as described in materials and methods. D: cells were treated for 6 or 48 h in isotonic or hypertonic conditions (150 mM extra of NaCl, 600 mosM total), and cell lysates were immunoblotted for the indicated protein. While MRTF expression does not show major changes at this osmolarity at either time, serum response factor expression (SRF) is affected.