FIG. 3.

Proteasome inhibition lengthens the circadian period in Rat-1 fibroblasts. (A to D) Rat-1 (mPer1::luc) fibroblasts were synchronized with 10 μM forskolin for 2 h. The synchronization medium was removed and replaced with medium containing the indicated protease inhibitors. (A) Synchronous luciferase oscillations between individual wells. Four individual wells used as the DMSO vehicle control were monitored for luciferase expression over time, and baseline-corrected data from each well was plotted. (B to D) Proteasome inhibition lengthens the circadian period. The indicated protease inhibitors were analyzed for their effect on period length. The average luciferase activity of cosynchronized DMSO controls is plotted in black, and the inhibitor is plotted in grey. Three to four wells were tested per compound and compared with the DMSO control experiment performed in the same 24-well plate. The experiments were repeated on at least four separate occasions with similar results. Each line is the baseline-corrected average for multiple wells from a single 24-well plate. Statistical significance was calculated by using the unpaired t test, and * and ** denote P values of <0.0025 and >0.5, respectively. Lact, lactacystin.