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1.
Fig. 6

Fig. 6. From: Acrolein Oxidizes the Cytosolic and Mitochondrial Thioredoxins in Human Endothelial Cells.

Effect of acrolein on HMEC-1 cell morphology. Cells with or without NAC pretreatment (2 mM for 2 hr) were treated with the indicated concentrations of acrolein for 30 min, washed, and incubated in fresh medium for 16 hr. The phase contrast images shown are representative of triplicate experiments.

Adam Szadkowski, et al. Toxicology. ;243(1-2):164-176.
2.
Fig. 7

Fig. 7. From: Acrolein Oxidizes the Cytosolic and Mitochondrial Thioredoxins in Human Endothelial Cells.

Quantification of the cytotoxic effects of acrolein on HMEC-1 cells and the partial protection afforded by NAC pretreatment. Cells with or without NAC pretreatment (2 mM for 2 hr) were exposed to the indicated concentrations of acrolein for 30 min, washed, and incubated in fresh medium without acrolein for 16 hr. From recorded phase contrast images (examples in Fig. 6), the percentage of cells that displayed visual signs of stress (e.g. rounded, condensed) were determined (mean ± S.D., n = 3). ** (p < 0.01) or *** (p <0.001) relative to the corresponding “No NAC” samples. # (p < 0.05) and ## (p < 0.001) relative to the 0 μM acrolein for “no NAC”.

Adam Szadkowski, et al. Toxicology. ;243(1-2):164-176.
3.
Fig. 3

Fig. 3. From: Acrolein Oxidizes the Cytosolic and Mitochondrial Thioredoxins in Human Endothelial Cells.

Redox status of Trx1 during acrolein-free recovery. HMEC-1 cells were treated with the indicated concentrations of acrolein for 30 min, washed in HBSS and incubated for the indicated times in MCDB131 medium with 10% FBS and supplements. At the end of the recovery time, the cells were washed once in HBSS and immediately processed for Trx1 redox status. The data (mean ± S.D. for 3 independent experiments) show the percentage of the total Trx1 that is in each of the three possible redox states: reduced (A), partially oxidized (B), and fully oxidized (C). * (p < 0.05), ** (p < 0.01), or *** (p <0.001) relative to the corresponding “No Recovery” samples for each acrolein concentration.

Adam Szadkowski, et al. Toxicology. ;243(1-2):164-176.
4.
Fig. 4

Fig. 4. From: Acrolein Oxidizes the Cytosolic and Mitochondrial Thioredoxins in Human Endothelial Cells.

The effect of geneticin on the redox status of Trx1 during a 4 hr acrolein-free recovery period. HMEC-1 cells were treated with the indicated concentrations of acrolein for 30 min, washed in HBSS and incubated for 4 hr in MCDB131 medium with 10% FBS and supplements, either with or without the protein synthesis inhibitor geneticin (800 μg per ml). At the end of the 4 hr period, the cells were washed once in HBSS and immediately processed for Trx1 redox status. The data (mean ± S.D. for 3 independent experiments) show the percentage of total Trx1 that is in each of the three possible redox states: reduced (A), partially oxidized (B), and fully oxidized (C). * (p < 0.05) or *** (p <0.001) relative to the corresponding 4 hr recovery samples without geneticin.

Adam Szadkowski, et al. Toxicology. ;243(1-2):164-176.
5.
Fig. 5

Fig. 5. From: Acrolein Oxidizes the Cytosolic and Mitochondrial Thioredoxins in Human Endothelial Cells.

The impact of NAC pretreatment on the redox status of Trx1. A: Redox western blot of Trx1 in HMEC-1 cells treated with the indicated concentrations of acrolein for 30 min. Prior to acrolein exposure, cells were either untreated (left) or pre-treated with 2 mM NAC for 2 hr in cell culture medium with FBS and supplements. The cells were then washed twice with HBSS and then treated with acrolein. Note that NAC-pretreated cells were not tested at 1 μM acrolein. B, C, D: The percentage of total Trx1 that is in the reduced (B), partially oxidized (C), or fully oxidized (D) form in cells with or without NAC pretreatment, as determined by densitometry of the redox blots (mean ± S.D. for 3 independent experiments). ** (p < 0.01) or *** (p <0.001) relative to the corresponding “No NAC” samples.

Adam Szadkowski, et al. Toxicology. ;243(1-2):164-176.
6.
Fig. 2

Fig. 2. From: Acrolein Oxidizes the Cytosolic and Mitochondrial Thioredoxins in Human Endothelial Cells.

Redox western blots of Trx2. A,B: HMEC-1 cells were either untreated or treated with the indicated concentrations of acrolein for 30 min, washed once in HBSS and immediately processed for Trx2 redox status. Standards for the migration of oxidized and reduced Trx2 are included, and consist of isolated mitochondria that had either been stored frozen (“mito, oxid.”) (Trx2 oxidizes in storage), or had been pre-treated with DTT which reduces Trx2 (“mito, DTT”). The AMS bonds to the –SH groups of the active site of reduced Trx2, increasing its mass. Reduced (AMS-adducted) Trx2 therefore migrates slower (Hansen et al., 2006a). Oxidized Trx2 migrated at 12 kDa consistent with its known mass. The blots were stripped and reprobed for GAPDH (36 kDa; loading control for cell samples). Relative to the cell lysates, only small amounts of the purified mitochondria were loaded so that the Trx2 band intensity better matched that of cell lysates; these lower mitochondrial protein loads are reflected in the weak GAPDH bands. The blots shown are representative of at least five independent experiments. C: Enlarged view of a Trx2 redox blot showing the 5 and 12.5 μM acrolein-treated cells.

Adam Szadkowski, et al. Toxicology. ;243(1-2):164-176.
7.
Fig. 1

Fig. 1. From: Acrolein Oxidizes the Cytosolic and Mitochondrial Thioredoxins in Human Endothelial Cells.

A: Redox western blot of Trx1 in HMEC-1 cells treated with the indicated concentrations of acrolein for 30 min, washed once in HBSS and immediately processed for Trx1 redox status. The 5 μM acrolein is equivalent to 10.7 fmol acrolein per cell. Iodoacetate covalently labels the –SH groups of reduced Trx1, and the resulting negative charge increases migration in native PAGE relative to oxidized Trx1. In partially oxidized Trx1 the active site C32/C35 is oxidized, whereas both dithiols (C32/C35, C62/C69) are oxidized in the fully oxidized form (Watson et al., 2003). A western blot of SDS-PAGE for GAPDH (loading control) for the same samples is shown. B: The relative distribution of the three redox states of Trx1 as determined by densitometry of redox blots. Points and error bars represent the mean ± S.D. (n = 3 independent experiments). For some, the error bars are smaller than the points as shown. Densitometry was not done on the 100 and 250 μM acrolein lanes because the bulk of material runs as a slower migrating smear rather than as distinct bands (see panel A).

Adam Szadkowski, et al. Toxicology. ;243(1-2):164-176.

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