BMDMs of HO-1^−/− mice did not survive EPC in vitro. (A) Bright-field pictures taken before and 1 hour after EPC showed no apparent difference between HO-1^−/− and HO-1^+/+ BMDMs. However, after 24 hours, WT macrophages returned to their normal pretreatment appearance, whereas HO-1^−/− macrophages were shrunken and did not regain their normal shape. (B) Measurements of cytosolic enzyme LDH in the culture medium showed that cells were equally intact in WT and HO-1^−/− samples 4 hours after EPC with the release of 12% of activity to the medium. At the 17- and 24-hour time points, LDH levels in the medium of HO-1^−/− cells were 51% and 59%, correspondingly 3- and 3.3-fold (P < .001) higher compared with WT. Error bars represent SD of the mean. (C) Quantitative RT-PCR demonstrated that HO-1 levels increased 5-, 37-, and 33-fold (P < .001) in WT cells 1, 3, and 6 hours after EPC, respectively (values are shown as ratios to actin-β). HO-1 expression returned to near the baseline levels 16 and 24 hours after treatment in WT cells. HO-2 levels did not increase in HO-1^−/− cells after EPC. (D) In WT FPN1, mRNA levels increased 11-fold 6 hours after EPC and subsequently decreased, showing a similar pattern to the HO-1 mRNA levels. This pattern of expression was distorted in HO-1^−/− BMDMs, with FPN1 levels 11-, 10-, 3-, and 3-fold higher at the 0-, 1-, 16-, and 24-hour time points, respectively (P < .001), than in WT cells. Different mice were used for different independent experiments where similar results were obtained. For each particular experiment, BM from either one WT or one HO-1^−/− mouse (age- and sex-matched) was used as a source of BMDMs.