Figure 5

CystC and Δ14CystC inhibit TGF-β signaling in human MDA-MB-231 breast cancer cells. (a) Control, CystC-expressing, or Δ14CystC-expressing MDA-MB-231 cells were transiently transfected with p3TP-luciferase and pCMV-β-Gal cDNAs, and were subsequently stimulated with increasing concentrations of transforming growth factor-β1 (TGF-β1; from 0 to 5 ng/ml) for 24 hours. Afterward, luciferase and β-Gal activities contained in detergent-solubilized cell extracts were measured. Values are luciferase activities (means ± SEM) observed in two independent experiments normalized to maximal reporter gene expression in TGF-β-stimulated cells expressing green fluorescent protein. CystC and Δ14CystC both significantly inhibit reporter gene expression stimulated by TGF-β (*P < 0.05; Student's t-test). (b) MDA-MB-231 cells were transiently transfected with p3TP-luciferase and pCMV-β-Gal cDNAs. Afterward, the transfectants were treated with 25 μg/ml recombinant glutathione S-transferase (GST; G), CystC (C), or Δ14CystC (D) and immediately stimulated with TGF-β1 (1 ng/ml) for 24 hours before determination of the luciferase and β-Gal activities contained in detergent-solubilized cell extracts. Values are luciferase activities (means ± SEM) observed in four independent experiments normalized to maximal reporter gene expression stimulated by TGF-β in GST-treated cells. (c) MDA-MB-231 cells were treated with 25 μg/ml recombinant GST (G), CystC (C), or Δ14CystC (D) for 2 hours before their stimulation with TGF-β (1 ng/ml) for 30 min. Afterward, the activation status of Smad2 was determined by immunoblot analysis with phospho-specific Smad2 antibodies. Differences in protein loading were monitored by reprobing stripped membranes with antibodies against extracellular signal-related kinase 1. Data are from a representative experiment that was repeated once with similar results.