Extended Data Figure 6 : CuET targets NPL4, causing immobilization and nuclear clustering of NPL4.

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

Extended Data Figure 6

a, CuET (1 μM) does not inhibit ATPase activity of p97. NMS873 (5 μM) was used as a positive control. Data are mean ± s.d. from four independent experiments. b, Western blotting analysis showing levels of ectopic p97–GFP, NPL4–GFP and UFD1–GFP in stable U2OS-derived cell lines used for the CuET-treatment rescue and cluster formation experiments. c, Ectopic expression of NPL4–GFP alleviates CuET-induced (125 nM, 4 h) accumulation of poly-Ub proteins in U2OS cells. d, Distribution of NPL4 nuclear clusters relative to chromatin in cells treated with CuET (1 μM, 2 h). Scale bars, 2 μm. e, Schematic representation of site-directed mutagenesis within the amino acid sequence of the putative zinc finger domain of NPL4. f, ITC curve showing the lack of CuET binding to purified NPL4(MUT) protein. g, DARTS analysis of recombinant NPL4 proteins shows that differential pronase-mediated proteolysis after CuET addition is apparent for NPL4(WT) but not for NPL4(MUT); detected by either silver-stained SDS–PAGE (the most prominent differential bands are marked by red dots) or by blotting with an anti-NPL4 polyclonal antibody. h, Viability of cells expressing doxycycline-inducible NPL4(MUT)–GFP, treated with CuET for 48 h. Data are from three independent experiments, means are linked. i, Accumulation of K48-ubiquitinated proteins and activation of UPR in cells expressing the doxycycline-inducible NPL4(MUT)–GFP. bd, f, g, i, Data are representative of two independent experiments.