Research Paper Volume 12, Issue 21 pp 21758—21776

Caspase-3 knockout attenuates radiation-induced tumor repopulation via impairing the ATM/p53/Cox-2/PGE2 pathway in non-small cell lung cancer

Figure 2. Casp3 KO attenuates radiation-induced apoptosis and growth-promoting effect of dying NSCLC cells. (A) Western blot analysis of the expression of caspase-3 in Casp3 KO A549 and H460 cells generated using the CRISPR/Cas9 system. β-tubulin was used as the loading control (***p<0.001, Student’s t test, n = 3). (B, C) The left panel shows the flow cytometry analysis of cell death in A549 and A549/Casp3 KO (B) and H460 and H460/Casp3 KO (C) cells following irradiation. Apoptotic cells were analyzed by Annexin V/propidium iodide (PI) double staining. The right panel shows the quantitative analysis of early apoptosis and total cell death in 0 Gy- or 8 Gy-irradiated control and A549/Casp3 KO (B) and H460/Casp3 KO (C) cells (***p<0.001, NS = not significant, Student’s t test, n = 3). (D) Casp3 KO significantly decreased the growth-promoting effect of 8 Gy-irradiated NSCLC cells on living NSCLC reporter cells. The upper panel depicts the luciferase activities showing the growth of A549 Fluc or H460 Fluc cells that were seeded with 8 Gy-irradiated wild-type or Casp3 KO cells or alone. The lower panel shows the representative bioluminescence images (***p<0.001, NS = not significant, one-way analysis of variance [ANOVA], n = 4).