Fig. 3: Ad-ChaC1 and AUR co-treatment leads to acute cellular stress responses. | Cell Death Discovery

Fig. 3: Ad-ChaC1 and AUR co-treatment leads to acute cellular stress responses.

From: ChaC1-based drug screenings identify a synergistic lethal effect of auranofin and proteasome inhibitors in hepatocellular carcinoma cells

Fig. 3

A, B Huh7 cells were infected with 1×108 pfu/mL Ad-NC, Ad-ChaC1 or Ad-ChaC1-Mut for 24 h. Cells were then treated with 0.3 µM AUR for 12 h. Reactive oxygen species levels were measured by DCFH-DA staining followed by flow cytometric analysis (A). Quantitative analysis was performed (B). C The expression levels of Nrf2, ATF3, ATF4 and ChaC1 were determined by western blotting. D Huh7 cells were infected with 1×108 pfu/mL Ad-ChaC1 for 24 h and then treated with 0.3 µM AUR for increasing time. The expression levels of Nrf2, ATF3, ATF4 and ChaC1 were determined by western blotting. E Proteomic profiling was conducted across six experimental conditions, including untreated controls, AUR monotherapy, Ad-NC infection alone, Ad-ChaC1 infection alone, Ad-NC + AUR co-treatment and Ad-ChaC1 + AUR co-treatment. Venn diagram analysis of differentially expressed proteins revealed 137 upregulated targets shared across Ad-ChaC1 and AUR co-treated groups. F The genes of interest (137 up-regulated genes) were input into DAVID Bioinformatics Resources (http://david.ncifcrf.gov). Analysis of Gene Ontology was shown. G Cells were treated as indicated. The protein expression levels of DDIT4, ATF4, ATF3, Nrf2 and ChaC1 were determined by western blotting. H Huh7 cells were infected with lentivirus expressing control shRNA or DDIT4-shRNA. The expression levels of DDIT4, ATF4, ATF3, Nrf2 and ChaC1 were determined by western blotting. I DDIT4-knockdown Huh7 cells and the control cells were infected with 1×108 pfu/mL Ad-NC or Ad-ChaC1 for 12 h, and then treated with 0.3 µM AUR for 24 h. Cells were stained with 1 µg/mL propidium iodide followed by flow cytometric analysis.

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