Fig. 5: Increased oxidative stress and cell death upon combined loss of BRCA1 and PALB2 in human cells.

a Levels of PALB2, NFκB p65 and phospho-NFκB p65 in control DAOY cells (PALB2-WT), PALB2 knockout DAOY cells (PALB2-KO), and the knockout cells reconstituted with a human PALB2 cDNA (PALB2-RC). A representative clone was used for each genotype. The PALB2-WT clone was a false positive PALB2-KO clone obtained after CRISPR-mediated genome editing. b ROS levels in the three cell lines in (a), as measured by the DCF assay. (c) Western blots showing levels of BRCA1, PALB2, NFκB p65, and phospho-NFκB p65 (S536) in the three cell lines after treatment with transfection reagent alone (no siRNA), a control siRNA, or two different siRNAs targeting BRCA1. d Relative ROS levels in the three cell lines treated with two different control or BRCA1 siRNAs. Data from each cell line were normalized separately, against the mean of the two control siRNAs. d, e Quantification of levels of NFκB p65 and phospho-NFκB p65 (S536) in the three cell lines after treatment with two different control or BRCA1 siRNAs. Data are normalized against the average of the two control siRNAs for PALB2-WT cells. g, h Apoptosis and necrosis of PALB2 knockout and reconstituted DAOY cells following depletion of BRCA1. Representative Annexin V assay results are shown in (g) and quantification in (h). At least three independent experiments were conducted for all quantifications. Error bars represent SD. *p < 0.05; **p < 0.01; ***p < 0.001.