Figure 2
Effects on VCP/p97 mRNA and protein expression, cross-resistance and changes in unfolded protein response (UPR) in resistant cell line. (a) Quantitative RT-PCR was performed to evaluate the mRNA expression of VCP in OVSAHO parental, O-CB-R1 and O-CB-R2. Bar graph represents the mean relative mRNA expression+S.E.M. normalized to 18 S from three independent experiments. P-values were calculated using Student’s t-test. (b) Whole-cell lysates were immunoblotted and probed with the antibody against VCP to evaluate the protein expression. β-Actin was used as the loading control. Numbers indicate relative VCP expression normalized to the β-actin loading control. (c) Table displays Mean GI50±S.E.M. (μM) with several VCP inhibitors and proteasome inhibitor (Bortezomib) from three independent experiments in parental and resistant cells. Numbers in the bracket for CB-5083 and NMS-873 treatment represent the drug resistance index (DRI) in resistant cells compared with parental cells. (d) OVSAHO parental, O-CB-R1 and O-CB-R2 were treated with CB-5083, DBeQ, bortezomib or tunicamycin at indicated concentrations. Cells were collected at 3 and 6 h time-points and whole-cell lysates were subjected to immunoblotting and probed with indicated antibodies. Numbers indicate relative ATF4 protein expression normalized to β-actin (loading control). **P value⩽0.01.
