Fig. 4

Generation and anti-proliferative activity of an antibody-drug conjugate targeting CD98hc. A Preparation of the antibody-drug conjugate targeting CD98hc. The coupling of DM1 to the anti-CD98hc antibody was analyzed by Western, by using an anti-DM1 antibody. Twenty nanograms of this ADC (αCD98hc-DM1), the nude anti-CD98hc (αCD98hc), trastuzumab or T-DM1 were loaded in 12% SDS-PAGE gels and analyzed for total protein (stain-free blot, lower image) and DM1 reactivity (upper panel). Trastuzumab and T-DM1 were used as a negative and positive controls. B Effect of anti-CD98hc-DM1 in a panel of TNBC cell lines. Cells were treated with anti-CD98hc and anti-CD98hc-DM1 10 nM for four days. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of triplicates of an experiment repeated twice. C Dose-response analyses of the effect of anti-CD98hc-DM1 on four TNBC cell lines. Cells were treated with the ADC for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of quadruplicates of an experiment repeated two times. D and E HS578T (D) and MDA-MB231 (E) cells were treated with anti-CD98hc, anti-CD98hc-DM1 or DM1 for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. F Knockout of CD98hc in MDA-MB231 cells by CRISPR/Cas9. Parental MDA-MB231 cells and two different clones knocked out for CD98hc were lysed. The levels of expression of CD98hc and LAT1 were analyzed by Western blot. Calnexin was used as a loading control. G Dose-response analyses of the effect of anti-CD98hc-DM1 on parental and CD98hc CRISPR #B3, #G3 MDA-MB231 cells. Cells were treated with anti-CD98hc-DM1 for four days. Results are shown as the mean ± SD of quadruplicates of an experiment repeated three times