Rnase a solution

H929 cells (1×10⁶ cells) treated with or without fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide or cytosine β-D-arabinofuranoside for 24 h at a concentration of 20 μg/mL were collected by centrifugation at 2,000 rpm for 5 min and washed once with PBS. The cell pellet was suspended in 70% ethanol for at least 4 h. Thereafter, the fixed cells were centrifuged at 2,000 rpm for 5 min, washed once with PBS, and then centrifuged at 2,000 rpm for 5 min. The cell pellet was suspended in 40 μL of phosphate-citrate buffer [0.2 M Na₂HPO₄, 0.1 M C₃H₄(OH)(COOH)₃: Sigma-Aldrich] and kept for 30 min at RT. After centrifuging at 2,000 rpm for 5 min, the cell pellet was suspended in 100 μL of PBS, and 1 μL of RNase A solution (10 mg/mL; Roche Diagnostics, Indianapolis, USA) was added and left for 30 min at 37°C. After centrifuging at 2,000 rpm for 5 min, the cell pellet was suspended in 1 mL of PBS, and 50 μL of PI solution (1.0 mg/mL; Sigma-Aldrich) was added and left for 30 min in the dark at RT. Finally, the stained cells were filtered through a cell strainer (BD Falcon; Bedford, MA, USA) and were analyzed using a fluorescence-activated cell sorter (FACS Calibur).

Mitochondria pellets from above were resuspended in TENTS buffer (100 mM Tris/HCl at pH 8.0, 50 mM EDTA, 0.5 M NaCl, 0.2% [v/v] Triton X-100; 1% [w/v] SDS) and incubated for 15 min at 60°C while shaking at 400 rpm. After adding 100 μl of a 10 mg/ml RNase A solution (50 U/mg; Roche Diagnostics GmbH, Manheim, Germany) samples were incubated for 1.5 h at 37°C. Subsequently, 100 μl of Proteinase K solution (10 mg/ml; Sigma-Aldrich, Steinheim, Germany) were added and samples placed over night at room temperature. Then, 630 μl phenol/chloroform/isoamyl alcohol (25:24:1) was added, probes incubated for 10 min at room temperature and then centrifuged at 18 000 × g for 10 min. After this, the supernatant was removed, 630 μl chloroform added and samples were centrifuged again at 18 000 × g for 10 min. Precipitation of mitochondrial DNA (mtDNA) was performed with 1/10 vol of 5 M NH₄-acetate and 1 vol isopropanol overnight at −20°C. After centrifugation for 45 min at 20 000 × g, the pellet was washed two times with 70% v/v and 100% v/v ethanol and re-suspended in 10 μl of 5 mM Tris/HCl, pH 7.6.