Purvalanol a

Confluent U2OS cell cultures grown in 150-mm dishes (2 × 10⁷) were treated with circadian period–altering compounds (longdaysin [cat. no. SML0127; Sigma-Aldrich], purvalanol A [cat. no. P4484; Sigma-Aldrich], roscovitine [cat. no. 557360; Calbiochem] and SP600125 [cat. no. S5567; Sigma-Aldrich]) to a final concentration of 10 μM dissolved in DMSO (100 μl of DMSO; <0.5% of the total culture volume) or an equivalent amount of DMSO (vehicle only). After 48-h incubation with the compounds or DMSO, the cells were harvested by trypsinization, washed twice in PBS, and were lysed in 500 μl lysis buffer containing 50 mM Hepes (pH 8.5), 8M urea, 1% NP-40, protease and phosphatase inhibitors, and benzonase nuclease. Then mild sonication was applied for 15 min (30 s on, 30 s off; medium power) using a Bioruptor Standard (Diagenode) instrument and lysates were centrifuged at 16,000g for 20 min at 4°C. Supernatants were carefully separated and transferred into new microcentrifuge tubes. Protein precipitation was performed with 1:6 volume of prechilled (−20°C) acetone overnight at 4°C. After overnight incubation, the lysates were centrifuged at 14,000g for 15 min at 4°C. Supernatants were discarded without disturbing pellets, and the pellets were air-dried for 2–3 min to remove residual acetone. Then the pellets were dissolved in 500 μl 100 mM triethylammonium bicarbonate (TEAB) buffer.

U2OS cells (American Tissue and Cell Collection, HTB-96) were a kind gift of Dr. John Hogenesh (University of Pennsylvania) NIH-3T3 (CRL-1658), MDCK (CCL-34) and 293T cells (CRL-3216) were directly obtained from ATCC. All cell lines were cultured in 10% Dulbecco’s modified Eagle medium supplemented to 10% decomplemented fetal bovine serum at 37°C, 5% CO₂, 21% O₂ and 100% humidity unless otherwise indicated. For aminoacid depletion experiments, dialyzed FBS was used and L-glutamine was supplemented as indicated. Cell lines were maintained and passaged according to ATCC recommended procedures. Pharmacological agents was as follows: Purvalanol-A (Sigma P4484), Z-VAD-FMK (EMD Millipore 627610), actinomycin D (Sigma A9415), cycloheximide (Sigma C7698), NAC (Sigma A9165), Trolox (Santa Cruz Biotech sc-200810) and CCCP (Sigma C2759) were added to cell cultures 12 h after cell seeding at the concentrations indicated and maintained until analysis, changing the medium every 24 hours. Ethynyl-uridine (Life Technologies E10345) and homopropargyl-glycine (Life Technologies C10186) were added at 5 and 50 μM respectively, two hours prior to incorporation analysis using the Click-iT alkyne detection kit (Life Technologies C10330).