Fig. 7

Fig. 7

Combination of VCR with afatinib or tariquidar induces apoptosis and reduces tumor volume in vivo. (A–D) Control BE(2)‐C and BE(2)‐C rVCR were treated with 40 ng·mL⁻¹ VCR, 2 μm afatinib, 100 nm tariquidar, or their combination for 48 h. all statistics were calculated with ANOVA of log‐transformed values followed by Tukey's post‐test. Shown are the statistics for the comparisons between the cell lines (bottom) as well as the comparisons to the solvent control for each cell line (top). (A, B) Western blot against PARP, BID, and transferrin receptor (TfR). Representative blots of three biological replicates are shown. Quantifications on the right depict biological replicates (n = 3) and their means. Fold changes depicted are cleaved PARP (cl‐PARP) over full‐length PARP (fl‐PARP), BID over GAPDH, and TfR over GAPDH, all normalized to the solvent control of each cell line. (C) Caspase‐3/7 activity was determined by cleavage of fluorescently labeled DEVD peptide in protein lysates. Depicted are biological replicates (n = 3) and their means, normalized to the solvent control of control BE(2)‐C. (D) Lipid peroxidation was evaluated by staining with 20 μm BODIPY 581/591 for 30 min followed by flow cytometric analysis of oxidized BODIPY. Depicted are biological replicates (n = 3) and their means normalized to the solvent control of control BE(2)‐C. (E) Change in tumor volume from day one post‐injection to day three post‐injection in zebrafish embryo xenografts with BE(2)‐C rVCR. Zebrafish embryos were treated with the indicated concentrations of VCR and tariquidar for 48 h. Each circle reflects one individual xenografts (solvent control n = 29; VCR n = 14; tariquidar n = 13, combo n = 19); means are presented by the black line. Statistics were calculated with Student's t test. ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant.