Rnases

DNA was isolated from the liver and lung tissues by a solvent extraction procedure [16] involving removal of RNA and proteins by digestion of isolated crude nuclei with RNases (Sigma Chemical Co., St. Louis, MO) and proteinase K (Roche Diagnostics, Indianapolis, IN), respectively, followed by sequential extractions with phenol (Amresco, Solon, OH), phenol:Sevag (1:1) and Sevag (choloroform:isoamyl alcohol; 24:1) (Fisher Scientific, Pittsburgh, PA). DNA was recovered by precipitation with ethanol, washed, dissolved in water and its concentration and purity was estimated by spectrophotometry.

A stock solution of ca. 1 mg/mL was first prepared by dissolving the lyophilized powder (0.26 mg) of RNAse S (Lot # 52H7034, Sigma-Aldrich, Steinheim, Germany) in 0.26 mL of 200 mM ammonium acetate, pH 7.0. Then, 100 µL were transferred onto a Microcon centrifuge filter with a 3 kDa cutoff (Millipore Corp., Bedford, MA, USA) together with further 200 µL of 200 mM ammonium acetate solution. This solution was centrifuged for 30 min at 13,000 rpm and 23 °C. The eluate was discarded and 200 µL of 200 mM ammonium acetate solution were added onto the filter. This procedure was repeated for three times. Then, the filter was inverted, placed into a new tube, and centrifuged for 5 min at 4500 rpm at 23 °C. A resulting supernatant of approximately 800 µL was collected and the protein concentration was determined to be 0.78 µg/µL using a Qubit^TM 2.0 Fluorometer (Carlsbad, CA, USA) assay. For nano electrospray mass spectrometry 2.56 µL of the purified and concentrated RNAse S solution were diluted to a final concentration of 0.2 µg/µL with 7.44 µL of 10% methanol/200 mM ammonium acetate. For each measurement, ca. 3 µL of the RNAse S solution were loaded into nanoESI capillaries using a microloader pipette tip (Eppendorf, Hamburg, Germany).

Type XII-A RNase A, RNase S, β-mercapto-ethanol, sodium phosphate (NaPi), and thioflavin-T (ThT) were purchased from Sigma-Aldrich. Ethanol (EtOH) and acetic acid (HAc) were obtained from Merck-Millipore. Yeast RNA was obtained from Boehringer. RNase S-protein was produced by precipitating the S-peptide from RNase S with EtOH [41 (link)]. Other chemicals were used at the highest purity available.

For cell cycle experiments, 10⁶ epithelial cells (MDBK or m-ICcL2) were placed in a 6-well plate. First, cells were treated overnight with SB203580 (25 μM). Cells were trypsinized, washed and fixed with 70% cold ethanol. After fixation, cells were treated with RNases (0.2 mg/ml, Sigma) for 1 h at 37°C, stained with propidium iodide (50 μg/ml) and analyzed by flow cytometry (Moflo Beckman Coulter, Fort Collins, Colorado, USA). The percentage of cells in each phase was assessed by using the software MultiCycle for windows Phoenix flow system, Inc. San-Diego, CA, USA.