Hx limits heme uptake, iron loading, and the production of ROS and cytokines in BMDM and Raw264.7 cells. (A) Heme levels in BMDMs treated with 5 μM Hx-heme or HSA-heme for 30 minutes (n = 3). (B) Heme levels in Raw264.7 cells treated with Hx-heme, HSA-heme, MSA-heme, or heme alone for 15, 30, or 60 minutes (n = 3). (C-H) HO-1, H- and L-ferritin, and FPN expression in Raw264.7 cells incubated with 5 μM Hx-heme, MSA-heme, or heme alone for the indicated times. In C and G, HO-1 and FPN mRNAs were analyzed by qRT-PCR (n = 4). Representative western blots are shown in D, E, F, and H (untreated, NT: n = 3; heme: n = 9). (I) Measurement of ROS levels in Raw264.7 cells treated with 10 μM Hx-heme, HSA-heme, or heme alone (6 and 15 hours; n = 4). (J-K) qRT-PCR analysis of TNFα and IL-6 mRNA expression in Raw264.7 cells treated with 5 μM Hx-heme or HSA-heme for 4 hours (n = 6). Values represent mean ± SEM. *P < .05; **P < .01; ***P < .001. mRNA levels measured by qRT-PCR are expressed in RQ to the untreated (NT) sample (RQ = 1); densitometric analysis is reported in AU, as the ratio to the untreated (NT) sample (AU = 1).