Extended Data Figure 4 : CuET inhibits the p97 pathway and induces cellular UPR.

From: Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4

Extended Data Figure 4

a, MG132-treated cells (5 μM, 6 h) accumulate both forms of NRF1 (120-kDa and 110-kDa bands, top and bottom arrows, respectively), whereas CuET-treated cells (1 μM, 6 h) accumulate only the non-cleaved 120-kDa form. b, Inhibition of the NRF1 cleavage process (appearance of the lower band) by CuET and NMS873 (a p97 inhibitor; 5 μM) in mouse NIH3T3 cells co-treated with the proteasome inhibitor MG132 (5 μM for 6 h). c, Time-course example images from a FRAP experiment, for which the quantitative analysis is shown in Fig. 2g (U2OS cells, blue boxes mark areas before bleaching, arrows after bleaching). d, U2OS cells pre-extracted with Triton X-100 and stained for poly-Ub(K48). The antibody signal intensities for cells treated with DMSO, BTZ (1 μM), NMS873 (10 μM) and CuET (1 μM) are analysed by microscopy-based cytometry and plotted below. e, Western blot analysis of accumulated poly-Ub proteins in the ultracentrifugation-separated microsomal fraction from U2OS cells treated with mock, CuET (1 μM), NMS873 (10 μM) or BTZ (1 μM) for 3 h. f, UPR in U2OS and MDA-MB-231 cell lines induced by 6-h treatment with CuET (various concentrations) or positive controls (5 μM NMS873, 2 μg ml−1 tunicamycin, 1 μM thapsigargin) is shown by increased levels of XBP1s, ATF4 and p-eIF2α. af, Data are representative of two independent experiments.