Figure 4 from Oncogenic Mutant <i>p53</i> Sensitizes Non–Small Cell Lung Cancer Cells to Proteasome Inhibition via Oxidative Stress–Dependent Induction of Mitochondrial Apoptosis

<p>The Onc-p53–NRF2–ATF3–NOXA pathway contributes to BTZ-induced apoptosis in <i>Onc-p53</i> NSCLC cells. <b>A,</b> H1975 cells were treated with vehicle or BTZ (10 nmol/L) ± NAC (1 mmol/L) for 24 hours, and confocal immunofluorescence microscopy was performed using NRF2 antibody (red) and DAPI to highlight nuclei (blue). Representative individual and merged images (40× magnification) of NRF2 and DAPI stains are shown. <b>B,</b> H1975 cells were treated with vehicle, BTZ, or BTZ + NAC, followed by NRF2 and DAPI staining as in <b>A</b> and fluorescence microscopy. The percentage of cells with NRF2 localization exclusive to the nucleus was determined from 10 random fields (>10 cells/field counted) at 20× magnification. A pairwise comparison was made using the Student <i>t</i> test between the two groups. <b>C,</b> H1975 cells were treated with vehicle, NAC (1 mmol/L), BTZ (5 nmol/L), or BTZ + NAC for 48 hours, and cell lysates were immunoblotted with NRF2 and GAPDH antibodies. <b>D,</b> H1975 cells stably expressing control shRNA (shC) or NRF2 shRNA (shNRF2) were treated with vehicle or BTZ (5 nmol/L) for 48 hours, and cell lysates were immunoblotted with indicated antibodies. <b>E,</b> H1975 shC and shNRF2 cells were treated with vehicle or the indicated concentrations of BTZ for 72 hours, and cell viability was determined by WST-1 assay. <b>F,</b> H1975-Control or H1975-p53KO cells were treated with vehicle (−) or BTZ (5 nmol/L) for 48 hours, followed by immunoblotting of cell lysates with indicated antibodies. **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001. Error bars indicate ± 1.0 SD.</p>

Duke Scholars

Author Eziafa I Oduah Medicine, Medical Oncology