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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Clin Cancer Res. 2019 Apr 2;25(15):4791–4807. doi: 10.1158/1078-0432.CCR-18-2751

Figure 5:

Figure 5:

Auranofin inhibited DUB, activated pro-apoptotic pathway of UPR and induced apoptosis in tumor cells. A) DUB activity in CT26 tumor cell lysates was measured by ELISA using the Ub-AMC substrate. Tumor tissue from mice treated with AUR+AIR showed lower DUB activity compared to irradiated (p<0.007) or untreated tumors (p<0.002). Treatment with auranofin without irradiation also reduced DUB activity in tumor tissue compared to irradiated (p<0.005) or untreated controls (p<0.002) (n=6 mice/group). B) Accumulation of ubiquitinated proteins was detected by western blot analysis with a monoclonal mouse primary antibody for ubiquitinated peptides. C) Histogram demonstrating mean band density of immunoreactive bands for ubiquitinated proteins. Combination treatment of auranofin and radiation significantly increased the ubiquitinated protein levels compared to irradiated (**p<0.005) or untreated control (*, p<0.001). Treatment with auranofin alone also increased ubiquitinated protein levels compared to untreated (*p<0.002) or irradiated control (**p<0.007). Representative western blot images from at least three independent experiments with β-actin as loading control (n=3 mice/group). D) qPCR analysis of sXBP1, GRP78, ATF4 and CHOP mRNA in CT26 tumor cells. There were several fold increases in sXBP1, GRP78, ATF4 and CHOP mRNA levels in the AUR+AIR treated group compared to AIR or auranofin treatment groups (n=6 mice/group). E-F) Immunoblot to detect p-PERK, PERK and CHOP expression in CT26 tumor cells. pPERK and CHOP expression were increased in the AUR+AIR treated group compared to control, AIR and auranofin treatment groups (n=3mice/group). G) Flow cytometry plot demonstrating Annexin V-FITC and PI positive apoptotic CT26 tumor cells. H) Histogram demonstrated that AUR+AIR treated tumor had higher numbers of Annexin V-FITC positive apoptotic cells compared to control (***,p<0.001) or AIR (**,p<0.003) or AUR treatment (*,p<0.007) (n=6mice/group). I) Representative images of CT26 colon tumor tissue showing TUNEL staining or immune-histochemical staining to detect peIF2alpha.