Suppressive effects of compound C on GRP78 and GRP94 protein accumulation.

<p>(A, B) Reporter assay. HT1080 cells were transfected with pGRP78pro160-Luc and exposed to stress (2DG, 10 mM 2-deoxy-D-glucose; TM, 5 µg/mL of tunicamycin (A) or GF, glucose-free (B)) for 18 h with compound C, versipelostatin or phenformin. Results shown are the means ± SD of quadruplicate determinations. (C–E) Immunoblot analysis. In C, HT1080 cells were treated for 18 h with compound C, versipelostatin or phenformin in the presence (+) or absence (−) of 10 mM 2DG. In D, 786-O cells (<i>left</i>) were treated for 18 h with compound C, versipelostatin or phenformin in the presence (+) or absence (−) of 10 mM 2DG. HeLa cells (<i>right</i>) were treated for 15 h with compound C in the presence (+) or absence (−) of 5 mM 2DG. Because HeLa cells exhibited hypersensitivity to cytocidal action of compound C during glucose deprivation, it was necessary to reduce the concentrations of 2DG and compound C, as compared with HT1080 and 786-O cell analysis. In E, HT1080 cells were treated for 18 h with compound C or versipelostatin in normal medium (Cont), normal medium with 10 mM 2DG (2DG) or glucose-free medium (GF). CC, compound C; VST, versipelostatin; Phen, phenformin. β-actin was used as a loading control. (F) MTT assay of HeLa cells treated with compound C under normal or ER stress conditions. 2DG, 10 mM 2-deoxyglucose; TM, 1 µg/mL of tunicamycin. Results shown are the means ± SD.