Figure 3 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>BTZ induces ATF3/NOXA and caspase-dependent apoptosis. <b>A,</b> H1975 cells were pretreated (30 minutes) with vehicle or the pan-caspase inhibitor QVD-OPh (QVD; 1 µmol/L) and then treated with vehicle or BTZ (5 nmol/L) for 48 hours. Cell viability was determined by trypan blue exclusion assay. <b>B,</b> H1975 cells were treated as in <b>A</b>, and the percentage of apoptotic cells was determined by annexin V–propidium iodide staining followed by FACS analysis. <b>C,</b> Cell lysates from <b>A</b> were immunoblotted with the indicated antibodies. <b>D,</b> H1975 or H460 cells were treated with vehicle or 2 or 5 nmol/L of BTZ for 48 hours, and cell lysates were immunoblotted with the indicated antibodies. <b>E,</b> RNA from <b>D</b> was extracted and subjected to qRT-PCR with <i>NOXA</i> primers. <b>F,</b> H1975 cells stably expressing shC or shNOXA were treated with vehicle or BTZ (5 nmol/L) for 48 hours, and cell lysates were immunoblotted with the indicated antibodies. <b>G,</b> H1975 cells were treated with vehicle or 2 or 5 nmol/L of BTZ for 48 hours, and mRNA expression of ATF3 was analyzed by qRT-PCR. <b>H,</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 indicated antibodies. <b>I,</b> H1975 cells stably expressing control shRNA (shC) or ATF3 shRNA (shATF3) were treated with vehicle or BTZ (5 nmol/L) for 48 hours, and cell lysates were immunoblotted with the indicated antibodies. <b>J,</b> H1975 cells stably expressing shC or shATF3 were treated with vehicle or BTZ (5 nmol/L) for 48 hours, and the percentage of apoptotic cells was determined by annexin V–propidium iodide staining followed by FACS analysis. **, <i>P</i> < 0.01; ***, <i>P</i> < 0.005; ns indicates <i>P</i> > 0.05. Error bars indicate ± 1.0 SD. Cl. Casp 3, cleaved caspase-3.</p>

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Author Eziafa I Oduah Medicine, Medical Oncology