Figure 2.

p97 P472L mutant cells are resistant to ATP-competitive and allosteric p97 inhibitors. A, P472L mutation was introduced into the p97 gene using homology-directed repair with CRISPR in HCT116 cells. Sequencing chromatograms from genomic DNA samples show incorporation of the missense C/T mutation to alter the Pro-472 codon and a silent A/G mutation that disrupts the targeted PAM in transfected and CB-5083–selected cell populations. B, P472L-edited HCT116 cell populations, control HCT116 cells, and NMS-873–resistant HCT116 cells harboring the A530T p97 mutation were tested in cell viability experiments. Inhibitors (2-fold serial dilutions from 5 μm CB-5083 or 20 μm NMS-873; 3-fold dilutions from 200 nm bortezomib) were added for 72 h (CB-5083 and NMS-873) or 48 h (bortezomib) prior to measuring ATP by luminescent detection. Resulting measurements were normalized to the maximum luminescence signal set at 100%. Data points represent the mean (n = 3) with S.D. IC₅₀ values with standard error (S.E.) from treatments that resulted in valid dose-response curves are indicated (ND, not determined). C, HCT116 and p97 P472L mutant cells were stably transfected with the p97 and proteasome substrate Ub–G76V–GFP and seeded to 96-well plates prior to incubating with CB-5083 (DMSO, 0.123, 0.37, and 1.1 μm) and NMS-873 (DMSO, 0.37, 1.1, and 3.3 μm) for 8 h. Integrated intensities (individual data points, mean, and, S.D. error) are shown. D, protein extracts from HCT116 and P472L cells treated with vehicle (DMSO) or inhibitors (0.3 or 2.5 μm, CB-5083; 2 or 5 μm, NMS-873) for 6 h were analyzed by Western blotting for ubiquitin, p97, ATF4, and CHOP.