Figure 3.

JNK activation is required for increased MS1 transcription during metabolic stress. (A) H9c2 cells were subjected to metabolic inhibition (MI) or allowed to recover (R) following 1h of metabolic inhibition for the times indicated. As a positive control, JNK was activated via osmotic shock, by exposure of the cells to 0.5 M sorbitol for 30 minutes (OS). Cell extracts were separated by SDS-PAGE and subjected to western blotting with antibodies reactive to phosphorylated (active) and total JNK as indicated. (B) H9c2 cells were preincubated without or with 25 µM SP600125, for 1 h and then subjected to metabolic inhibition (MI) or allowed to recover (R) following 1h of metabolic inhibition in the continued presence of the JNK inhibitor. MS1 and β-actin mRNA levels were determined by RT-PCR and MS1 normalised against β-actin. Results are the mean ± SEM (n = 3). (C) In control experiments, serum-starved H9c2 cells were preincubated with 25 µM SP600125 for 1 h prior to incubation with 10% serum, to activate JNK. Cell lysates were then blotted for phospho-c-Jun and tubulin.