Effects of mechanical stress on c-Fos expression. mRNA levels were analyzed by quantitative real time RT-PCR in HC11 cells stretched for 1 h using different strain intensities (A) or subjected to 20% RSA for the indicated time periods (B); gapdh expression was used to normalize c-fos mRNA levels. c-Fos protein levels were analyzed by western blot in HC11 cells subjected to 20% RSA for the indicated times (C). Nuclear fractions from HC11 mammary epithelial cells were analyzed by western blot to determine c-Fos protein presence in this compartment after 3 h of sustained 20% RSA (D). Analysis was performed by triplicate in at least three independent experiments. Significant differences: (*) p < 0.05 compared to 0% RSA and (**) p < 0.05 compared to 5% RSA.