Figure 1
From: Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress
Cell culture model of heme toxicity. (a) Hmox1 (+/+) and Hmox1 (−/−) cells were incubated with 10 μM heme for 4 h. The relative Hmox1 mRNA induction in response to heme exposure was quantified by TaqMan RT-PCR. The mRNA levels are indicated as fold expression over control. No transcript was detected in the Hmox1 (−/−) cell line. (b) Cell pellets of Hmox1 (+/+) and Hmox1 (−/−) cells after 24 h of heme exposure (10 μM). Before imaging, cells were washed three times in large volumes of PBS containing EDTA. (c and d) Hmox1 (+/+) MEFs (blue) and Hmox1 (−/−) MEFs (red) were treated with the indicated concentrations of heme for 12 h. Intracellular ATP (c) and caspase 3/7 activity (d) were measured by luminescence assays. Data represent the mean±S.D. of six biologic replicates. (e) Hmox1 (+/+) MEFs (blue) and Hmox1 (−/−) MEFs (red) were treated with the indicated concentrations of heme for 14 h. Condensed nuclei were quantified by digital image analysis after Hoechst staining. Data include images (15 per replicate) from three independent biologic replicates. (f) Hmox 1 (−/−) MEFs were incubated with or without 20 μM heme for 12 h and stained with Hoechst (nuclei=yellow) and Alexa 488 phalloidin (actin cytoskeleton=cyan). The heme toxicity, which was characterized by cytoskeleton rarefication and nuclear condensation, was totally prevented by hemopexin (Hpx; 0.5 mg/ml). Images were acquired with a Zeiss Observer Z1 equipped with an ApoTome.2 module and an Axiocam MRm at × 400 original magnification. °indicates outlier values
