Figure 6

Bucillamine increases expression of γ-GCS and GSS in HEI-OC1 cells. (a) Cells were pretreated with 2 mM bucillamine for 1 h followed by the addition of 20 μM cisplatin for different time intervals as indicated. Total RNA was isolated and cDNA was synthesized by reverse transcription. cDNAs of γ-glutamylcysteine synthetase (γ-GCS: GCLC and GCLM) and glutathione synthetase (GSS) were amplified using specific primer sets. (b) mRNA expression levels of GSS, GCLC and GCLM were analyzed by quantitative real-time PCR at 12 h. (c) Cell lysates were collected, electrophoresed through an SDS-polyacrylamide gel, and subject to immunoblot analysis with antibodies against γ-GCS (GCLC and GCLM) or GSS. The protein and mRNA levels of β-actin were used as controls. (d) Intracellular GSH content was measured by using a Glutathione Assay Kit. Total GSH contents were expressed as nmol mg⁻¹ protein. Values are represented as the mean (s.d.) of triplicate samples. *P<0.01 compared with control.