exposure, wash and fresh medium was repeated Monday through Thursday for one pair of samples and Tuesday through Friday for a second. After washing the cells on Thursday (or Friday for the second pair), all dishes were split and plated in medium without ethanol until the following Monday. This pattern was repeated in week 2. In week 3, the treated group was split into two flasks on Monday (or Tuesday) and both were treated with ethanol as above for 3 days but on Thursday (or Friday), one group was treated with 40 mM ethanol for 4 h, the other received medium without ethanol. All cells were collected after 4 h. RNA was extracted using a Direct-zol DNA/RNA miniprep kit (R2080, Zymo Research, www.zymoresearch.com) per manufacturer’s instructions. The repeated washings and media changes removed most non-adherent cells, so the cells being studied were primarily adherent. Six independent biological replicates were conducted for each condition, with each pair of samples starting in on a new day (Monday or Tuesday of 3 successive weeks). The experiment thus spanned 5 weeks, during which cells were from passages 6 to 14 after receipt from ATCC.
