Article Title: Alcohol-abuse drug disulfiram targets cancer via p97 segregase adapter NPL4
Figure Lengend Snippet: CuET inhibits the p97 pathway and induces cellular unfolded protein response (UPR) a ) Proteasome inhibitor MG132-treated cells (5μM, 6h) accumulate both forms of NRF1 (120- and 110-KDa bands, upper and lower arrows, respectively) while CuET-treated cells (1μM, 6h) 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; 5μM) in mouse NIH3T3 cells co-treated with the proteasome inhibitor MG132 (5μM for 6h); c ) Time-course example images from a FRAP experiment the quantitative analysis of which is shown in Fig. 2g (U-2OS cells, blue boxes mark areas before bleaching, arrows after bleaching); d ) U-2OS cells pre-extracted by TritonX and stained for K-48-polyUb. The Ab signal intensities for cells treated with DMSO, BTZ (1μM), NMS873 (10μM) and CuET (1μM) are analysed by microscopy-based cytometry and plotted below; e ) Western blot analysis of accumulated poly-Ub proteins in ultracentrifugation-separated microsomal fraction from U-2OS cells treated by mock, CuET (1μM), NMS873 (10μM) or BTZ (1μM) for 3h; f ) UPR in U-2OS and MDA-MB-231 cell lines induced by 6-h treatment with CuET (various concentrations) or positive controls (NMS873 5μM, tunicamycin 2μg/ml, thapsigargin 1μM) manifested by increased levels of Xbp1s, ATF4 and p-eIF2a. Panels a-f are representative of two independent experiments.
Article Snippet: :3933), anti-H2A, acidic patch (1:1000; Merck Millipore, cat. n.: 07-146), anti-monoubiquityl-H2A (1:1000; Merck Millipore, clone E6C5), anti-IκBα (1:500; Santa Cruz Biotechnology, cat. n.: sc-371), anti-p53 (1:500; Santa Cruz Biotechnology, clone DO-1), anti-HIF1α (1:1000; BD Biosciences, cat. n.: 610958), anti-Cdc25A (1:500; Santa Cruz Biotechnology, clone DCS-120), anti-NRF1 (1:1000, Cell Signaling, clone D5B10), anti-VCP (1:2000; Abcam, cat. n.: ab11433), anti-VCP (1:1000; Novus Bio, cat. n.: NBP100-1557), anti-NPLOC4 (1:1000; Novus Bio, cat. n.: NBP1-82166), anti-ubiquitin lys48-specific (1:1000; Merck Millipore, clone Apu2), anti-β-actin (1:2000; Santa Cruz Biotechnology, cat. n.: sc-1616; or 1:500, Santa Cruz Biotechnology, cat. n. sc-87778), anti-GAPDH (1:1000,GeneTex, clone 1D4), anti-Lamin B (1:1000; Santa Cruz Biotechnology, cat. n.: sc-6217), anti-calnexin (1:500; Santa Cruz Biotechnology, cat. n.: sc-11397), anti-α-Tubulin (1:500; Santa Cruz Biotechnology, cat. n.: sc-5286), anti-Xbp1 (1:500; Santa Cruz Biotechnology, cat. n.: sc-7160), Ufd1 (1:500; Abcam, cat. n.: ab155003), cleaved PARP1 (1:500; Cell Signaling, cat. n.: 9544), p-eIF2a (1:500; Cell Signaling, cat. n.: 3597), ATF4 (1:500; Merck Millipore, cat. n.: ABE387), HSP90 (1.500; Enzo, cat. n.: ADI-SPA-810), HSP70 (1:500; Enzo, cat. n.: ADI-SPA-830), HSF1(1:500; Cell Signaling, cat. n.: 4356), pHSP27 (1:1000; Abcam, cat. n.: 155987), HSP27 (1:1000; Abcam, cat. n.: 109376) followed by detection by secondary antibodies: goat-anti mouse IgG-HRP (GE Healthcare), goat-anti rabbit (GE Healthcare), donkey-anti goat IgG-HRP (Santa Cruz Biotechnology, sc-2020).
Techniques: Inhibition, Staining, Microscopy, Cytometry, Western Blot, Multiple Displacement Amplification