1. Selective Peroxidation and Externalization of Phosphatidylserine in Normal Human Epidermal Keratinocytes During Oxidative Stress Induced by Cumene Hydroperoxide 
Anna A. Shvedova, Julia Y. Tyurina, Kazuaki Kawai, Vladimir A. Tyurin, Choudari Kommineni, Vincent Castranova, James P. Fabisiak, Valerian E. Kagan 
Journal of Investigative Dermatology 
Volume 118, Issue 6, Pages (June 2002)
DOI: /j x
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Effect of Cum-OOH on oxidation of PnA-labeled phospholipids in live NHEK and liposomes prepared from phospholipids of NHEK. NHEK were labeled with PnA and then incubated in phenol red-free KGM-2 basal medium in the presence or absence of Cum-OOH (50–200 µM) for 1 h at 37°C. At the end of incubation, NHEK were harvested by trypsinization, washed twice with PBS, and lipids were extracted and resolved by HPLC as described in Materials and Methods. Closed circles, NHEK; open circles, liposomes prepared from NHEK prelabeled with PnA. Data represent mean ± SEM obtained from five experiments. *p < 0.01; **p < 0.005, ***p < 0.001 vs control NHEK (Cum-OOH nontreated); #p < 0.05 vs phosphatidylethanolamine or phosphatidylcholine from NHEK treated with 200 µM Cum-OOH. PI, phosphatidylinositol; PE, phosphatidylethanolamine, PS, phosphatidylserine, PC, phosphatidylcholine.
Journal of Investigative Dermatology  , DOI: ( /j x)
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3. Figure 2
HPTLC analysis of total and fluorescamine-modified phospholipids from control and Cum-OOH-exposed NHEK. Total lipid extracts from control (A, B) and Cum-OOH-treated NHEK (C, D) were subjected to two-dimensional HPTLC. Identities of lipids are: NL, neutral lipids; FFA, free fatty acids; PEA, phosphatidylethanolamine; phosphatidylcholine, phosphatidylcholine; Sph, sphingomyelin; PI, phosphatidylinositol; PS, phosphatidylserine; Dpg, diphosphatidylglycerol; mPEA, fluorescamine-modified phosphatidylethanolamine; mPS, fluorescamine-modified phosphatidylserine. The chromatograms were visualized using a Bio-Rad Fluor-S Multiimager equipped with a UV vis light source. (A, C) Fluorescence of fluorescamine-modified lipids. (B, D) Total lipids after exposure to iodine vapors.
Journal of Investigative Dermatology  , DOI: ( /j x)
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4. Figure 3
Effect of Cum-OOH on externalization of phosphatidylserine in NHEK. NHEK were incubated in phenol red-free KGM-2 basal medium in the presence or absence of Cum-OOH (50 or 200 µM) for 1 h at 37°C. After the end of incubation, NHEK were treated with fluorescamine for 30 s, washed, harvested by trypsinization, and lipid were extracted and resolved by HPTLC as described in Materials and Methods. Panel A shows the amount of derivatized phosphatidylethanolamine (PE) and panel B shows the amount of derivatized phosphatidylserine (PS). Data are expressed as a percent of the total (unmodified + modified) pool of that particular phospholipid. Data represent mean ± SEM obtained from three experiments. *p < 0.05 vs control (NHEK nontreated with Cum-OOH).
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Nuclear morphology of normal and Cum-OOH-exposed NHEK. NHEK were incubated in phenol red-free KGM-2 basal medium in the absence (A) or in the presence (B) of Cum-OOH (200 µM) for 1 h at 37°C. Following incubation Cum-OOH was washed out and NHEK were incubated in fresh KGM-2 basal medium for an additional 5 h at 37°C. NHEK were then harvested by trypsinization, washed with PBS, fixed with 2% paraformaldehyde in PBS, and stained with Hoechst Photomicrograph is representative of three experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
Electron microscopy of NHEK treated with Cum-OOH. Typical electron micrographs of control NHEK (A) and NHEK treated with 200 µM Cum-OOH for 2 h (B) are shown. No morphologic distinctions in the nucleus or cytoplasm were evident between the Cum-OOH exposed and control NHEK. View of normal ultrastructural appearance of nucleus and nucleolus. Original magnification × 6750. Conditions: NHEK (5 × 106) were treated with. Cum-OOH-treated and control cells were fixed immediately after exposures to 200 µM Cum-OOH or vehicle, respectively, at 37°C in CO2 incubator in phenol red-free KGM-2 basal medium for 2 h.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Effect of Cum-OOH on caspase-3 activity in NHEK. NHEK were incubated in phenol red-free KGM-2 basal medium in the presence or absence of Cum-OOH (50 or 200 µM) for 1 h at 37°C. After that, Cum-OOH was washed out and NHEK were incubated in fresh KGM-2 basal medium for 5 h at 37°C. NHEK were then harvested by trypsinization and activity of caspase-3 was determined as described in Materials and Methods. *p < 0.05 vs control (NHEK nontreated with Cum-OOH). Data represent mean ± SEM obtained from three experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
Viability of NHEK following exposure to Cum-OOH. Cells were exposed to Cum-OOH for 1 h and then media changed to fresh media without Cum-OOH. Viability was assessed by Trypan Blue at indicated times after exposure. Data represent mean ± SEM obtained from four experiments. *p < 0.05 vs control.
Journal of Investigative Dermatology  , DOI: ( /j x)
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