Journal: Theranostics

Article Title: CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells

doi: 10.7150/thno.72826

Figure Lengend Snippet: USP1 interacts with and regulates the MAST1 protein. (A) Schematic representation of the sgRNAs targeting exon 5 of the USP1 gene. Red arrowheads indicate the positions of sgRNAs that target the top strand. sgRNA sequences are in red; PAM sequences are in bold blue font. (B) Validation of sgRNA efficiency targeting USP1 by transient transfection of sgRNA1 and sgRNA2 into HeLa cells and immunoblotting with USP1 antibody. (C) HeLa cells were transfected with sgRNA1 and shRNA1 targeting USP1 , and the endogenous protein levels of USP1 and MAST1 were checked by Western blotting. (D) HeLa cells were transfected with increasing concentrations of Flag-USP1 to check the endogenous MAST1 protein level. (E) HeLa cells were transfected with increasing concentrations of Flag-USP1CS to assess the endogenous MAST1 protein level. (F) The reconstitution effect of Flag-USP1 on endogenous MAST1 protein in USP1-depleted HeLa cells. The protein band intensities (Fig C-F) were estimated using ImageJ software with reference to the GAPDH control band for each individual sgRNA (MAST1/GAPDH) and presented below the blot. (G) Interactions between endogenous and (H) exogenous USP1 and MAST1 proteins were analyzed in HeLa cells and HEK293 cells, respectively. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. Protein expression was checked using Western blotting. GAPDH was used as a loading control. (I) HeLa cells were subjected to the Duolink PLA assay to analyze the interaction between USP1 and MAST1 using specific antibodies. In situ USP1-MAST1 interaction (PLA dots) was observed when USP1 and MAST1 were immunostained together but not when they were stained with individual antibodies. Scale bar: 10 µm. (J) HeLa cells were treated with either MLN7243 (10 µM) or PR-619 (20 µM) for 1 h before harvesting. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (K) Schematic representation of full length USP1 (1-785 aa) encoding USP domain with catalytic triad residues (Cys, His, and Asp box) (represented as USP1-WT), N-terminus USP1 (1-400 aa) encoding catalytic Cys box (represented as USP1-UTM1), C-terminus USP1 (401-785 aa) encoding catalytic His and Asp box (represented as USP1-UTM2), and extended C-terminus USP1 (201-785 aa) encoding catalytic His and Asp box (represented as USP1-UTM3). Interactions between full length MAST1 and USP1 truncated mutants by co-immunoprecipitation and immunoblotting with the indicated antibodies (lower panel). (L) Schematic representation of full length MAST1 (1-1570 aa) encoding serine/threonine (S/T) kinase domain and PDZ domain (represented as MAST1-WT), N-terminus MAST1 (1-832 aa) encoding S/T kinase domain (represented as MAST1-MTM1), C-terminus MAST1 (833-1570 aa) encoding PDZ domain (represented as MAST1-MTM2), and C-terminus MAST1 (1118-1465 aa) lacking PDZ domain (represented as MAST1-MTM3). Interactions between full length USP1 and MAST1 truncated mutants were analyzed by co-immunoprecipitation and immunoblotting with the indicated antibodies (lower panel).

Article Snippet: Protein A/G Plus agarose beads (sc-2003, Santa Cruz Biotech), protease inhibitor cocktail (Cat. no. 11836153001, Roche), IP lysis buffer (Cat. no. 87787; Thermo Fisher), cell lysis buffer (Cat. no. R2002, Biosesang), protein 5X sample buffer (Cat. no. EBA-1052, Elpis Biotech), protein translation inhibitor cycloheximide (CHX; Cat. no. 239765, Merck), proteasomal inhibitor MG132 (Cat. no. S2619, Selleckchem), puromycin (Cat. no. 12122530, Gibco), cisplatin (Cat no. P4394, Sigma-Aldrich), pimozide (Cat no. P1793, Sigma-Aldrich), lestaurtinib (Cat no. 3395, Tocris), DUB inhibitor, PR-619 (ab144641, Abcam), ubiquitin activating enzyme inhibitor, MLN7243 (also called TAK243, Cat no. HY-100487, MedChemExpress) and DAPI (Cat. no. H-1200, Vector Laboratories) were also used.

Techniques: Transfection, Western Blot, Software, Control, Immunoprecipitation, Expressing, In Situ, Staining