Figure 8.

Effect of H₂S-treated albumin on PX-12-induced cell death. (A) Induction of the reduced form of albumin by H₂S. Albumin (12.5 μg) pretreated with or without 1mM NaHS were exposed to 250 μM PX-12 followed by reaction with thiol-reactive MAL for 2 h. and the signal of MAL-labeled albumin was detected using dot blot. Densitometric analysis of the blot in (A) was shown in (B). Data shown are mean ± SE, (n = 4; ^**P < 0.01 vs. control; ^##P < 0.01 vs. PX-12 alone). (C) H₂S induced enhancement of sulfhydryl residues and sulfhydration in albumin. Albumin (12.5 μg) treated with or without 1 mM NaHS was assayed for sulfhydration as described in the section of Materials and Methods. The samples were added to dot-blot apparatus and detected for the fluorescent signal. To confirm the equal loading of albumin, the membrane was stained with EZ blue (C, input). Note the obvious increased MAL-labeled Trx after NaHS treatment in comparison with control and its reduction following DTT treatment. (D) Densitometric analysis of the intensity of bands between control and NaHS-treated samples without DTT treatment. (E) Quantitative calculation of sulfhydrated albumin by NaHS. The level of sulfhydration was calculated through the fluorescence loss after DTT treatment and expressed as a percentage against the intensity of fluorescence before DTT treatment. Data in (D,E) are representative of four independent experiments and values are expressed in mean ± SE, (^*P < 0.05, ^**P < 0.01 vs. control). (F,G) Effect of NaHS-treated albumin on PX-12-induced cell death. HepG2 cells in 96-well plate were exposed to the indicated concentrations of PX-12 in the presence or absence of 20 mg/ml NaHS-treated albumin for 4 h. Then, the cells were stained with Calcein-AM/PI (F, magnification: × 400). Cell viability was determined by WST assay (G). Data in (G) are expressed as the percentage of living cells against the respective control (mean ± SE, n = 4; ^**P < 0.01 vs. control; ^##P < 0.01 vs. PX-12 alone).