Figure 2. Effects of clinically relevant hypothermia and hyperthermia on cell levels and distribution of TCF7 and β-Catenin.

A. SAECs were incubated at 32°C, 37°C or 39.5°C for 24h and whole cell lysate was immunoblotted for β-Catenin, TCF7, and ß-tubulin. The band densities were quantified, normalized to ß-tubulin band density, and expressed as fold-change vs. 37°C. A representative immunoblot and a graph summarizing the mean±SE of 4 experiments is shown. B. SAECs grown on chamber slides were incubated at 32°C, 37°C or 39.5°C for 24h and either fixed (left panels) or switched back to 37°C and stimulated with 200 ng/ml Wnt-3a for 3h and fixed. The fixed monolayers were immunostained for β-Catenin (red) and DAPI (blue) and analyzed by confocal immunofluorescence microscopy. Representative fields from three similar experiments are shown. C. HEK293T cells were transfected with either inhibitors (inh) or mimics (mim) of the three temperature responsive miRNAs (miR x 3), scrambled sequence controls for each (cnt) or nothing (−), recovered for 24 h, incubated at the indicated temperature for 24 h, then stimulated with 200 ng/ml Wnt-3a at 37°C for 3h and Axin2 mRNA levels were measured by qRT-PCR and expressed as fold-change vs. unstimulated 37°C cells. Mean±SE of 3 experiments. * and † denote p<0.05 vs. unstimulated 39.5°C and mimic-pretreated cells, respectively.