Figure 5:

USP22 modulates PALB2 protein stability aiding in chemoresistance. a. Western blot of H1299 cells against endogenous USP22, BRCA2, PALB2, Rad51, and β-Actin (loading control) after 48-hour siRNA knockdown. Numbers to left of western blots indicate molecular weight. b. U2OS^FOKI cells treated with 4OHT and SHIELD1 ligand to induce DDR at the 256X LacO array by the endonuclease mCherry-LacI-FOKI^WT with/without USP22 siRNA knockdown for 48 hours and/or transfection of empty vector or FLAG-USP22 plasmids. Cells were stained with antibodies against indicated proteins. Percentage of cells with recruitment of indicated protein to the array are indicated with +/−SD. White scale bars indicate 5um. 120 cells were counted for each condition for every experimental round. Experiment was done in triplicate. c. Western blot against endogenous USP22 and β-Actin (loading control) after 48-hour siRNA knockdown, refers to cells from 5b. Numbers to left of western blots indicate molecular weight. (Right) western blot to deduce expression of indicated FLAG-USP22 construct. Western blot done against FLAG and β-Actin (loading control). Numbers to left of western blots indicate molecular weight. d. H1299 or H1299^FLAG-PALB2 cells stably overexpressing indicated protein had siRNA KD performed then 3uM/6uM cisplatin or DMSO was added in for an additional 72 hours. Cells were washed then counted. Graph indicates total % of living cells as they were pertaining to the control for both cell lines. Experiment was done in triplicate. e. Western blot for cells from Figure 5d against endogenous USP22, PALB2, and β-Actin (loading control). FLAG antibody was used to assay for FLAG-PALB2. Numbers to left of western blots indicate molecular weight.