TRTS-induced changes in PC12 cell differentiation after exposure to various inhibitors.

<p>(A–K) PC12 cells in the differentiation medium were treated with TRTS at 39.5°C (18 h/day), 40 ng/ml BMP4, or left untreated for 7 days in the presence or absence of 2.5 μM U0126, 2.5 μM U0124, 0.5 μM SB203580, or 1.0 μM GW441756. (A) Representative phase-contrast images of PC12 cells on day 7 after treatment with no stimulation; (B) TRTS (18 h/day); (C) TRTS (18 h/day) plus 2.5 μM U0126; (D) TRTS (18 h/day) plus 2.5 μM U0124; (E) TRTS (18 h/day) plus 0.5 μM SB203580; (F) TRTS (18 h/day) plus 1.0 μM GW441756; (G) 40 ng/ml BMP4; (H) 40 ng/ml BMP4 plus 2.5 μM U0126; (I) 40 ng/ml BMP4 plus 2.5 μM U0124; (J) 40 ng/ml BMP4 plus 0.5 μM SB203580; (K) 40 ng/ml BMP4 plus 1.0 μM GW441756. Scale bar, 100 μm. (L, M) PC12 cells were treated with TRTS at 39.5°C (18 h/day), 40 ng/ml BMP4, or left untreated for 7 days in the presence of the indicated concentrations of U0126, U0124, SB203580, or GW441756. The percentage of neurite-bearing cells on day 7 was assayed. The data represent the mean ± SD of three replicates. ††<i>P</i> < 0.01 vs. the day 7 control with no stimulation. **<i>P</i> < 0.01 vs. TRTS or BMP4 alone on day 7. n.s., not significant. #, not significant vs. the day 7 control with no stimulation. TRTS, temperature-controlled repeated thermal stimulation.