Fig 2: SprT domain of SPRTN and USP domain of USP11 mediate SPRTN-USP11 interaction.A, Top panel, protein schematic of SPRTN FL, domain deletion, and point mutants. Bottom panel, HEK 293T cells were transfected with Myc-USP11 and SFB-SPRTN FL, deletion, or point mutants. B, Top panel, schematic of USP11 FL and deletion mutants. USP11 domains derived from Pfam predicted motifs. Bottom panel, HEK 293T cells were transfected with SFB-SPRTN and Myc-USP11 FL, deletion, or point mutants. C, Left panel, protein schematic of USP11 FL and USP domain deletion mutants. Right panel, HEK 293T cells were transfected with SFB-SPRTN and Myc-USP11 FL or USP domain deletion mutants. D, Left panel, protein schematic of SPRTN FL and SprT domain deletion mutants. Right panel, HEK 293T cells were transfected with SFB-SPRTN FL and SprT domain deletion mutants. A–D, cell lysates were immunoprecipitated with S-protein agarose beads, and immunoblotting was performed using the indicated antibodies. WB, western blot. ∗∗∗ indicates SPRTN auto-cleavage products.

Fig 3: Catalytic activity of USP11 is essential to regulating XPC deubiquitination and NER after UVB exposure(A) Immunoblot analysis of XPC, myc, and GAPDH in 293T ΔUSP11 cells transfected with or without myc- tagged wild-type (WT) and catalytic mutant USP11 plasmids (csmt USP11) at the indicated times post-UVB (20 mJ/cm2) or -sham irradiation. (B) Slot blot analysis of the CPD levels at indicated times after UVB (5 mJ/cm2) in 293T ΔUSP11 cells transfected with WT USP11 or csmt inactive mutant USP11 plasmids. (C) Quantification of percentage (%) of CPD repair from (B) (mean ± S.E., n=3). *, P < 0.05, compared with WT USP11 group, Student’s t-test.

Fig 4: USP11 as a deubiquitinase stabilizes XIAP from ubiquitin-dependent degradation.(A) While elevated expression of USP11-WT led to increased XIAP protein levels, expression of deubiquitinase catalytic death mutant USP11 failed to stabilize XIAP in MCF10A cells. (B) Expression of USP11-WT but not USP11-C318A stabilizes Myc tagged XIAP expression levels in HEK293 cells. (C) Depletion of USP11 leads to decreased turnover half-life of XIAP in MDA-MB-231 cells. The density of XIAP band was quantified, normalized to the internal control β-actin, which are presented in right panel. (D) Elevated expression of USP11 results in prolonged XIAP protein turnover half-life in MCF10A. The density of XIAP band was quantified, normalized to the internal control β-actin, which are presented in right panel. (E) Blockade of USP11 by pharmacological inhibitor Mitoxantrone destabilizes XIAP protein in MDA-MB-231 cells. MDA-MB-231 cells were treated with Mitoxantrone at indicated concentration for 24 h. (F) Expression of USP11-WT but not USP11-C318A results in decreased XIAP ubiquitin-conjugates in HEK293 cell. HEK293T cells were transfected with the indicated plasmids for 48 h and then collected for immunoprecipitation assay with the FLAG tag antibody followed by the immunoblotting analysis with the HA and Myc tag antibody. (G and H) Elevated expression of USP11 and XIAP are significantly correlated in 65 cases human breast cancer tissue specimen. G Represents paired immunohistochemistry staining of USP11 and XIAP. H Shows statistical analysis of immunohistochemistry staining and indicates USP11 expression is positively correlated with XIAP expression in breast cancer (R = 0.434, p < 0.001).

Fig 5: Identification of USP11 as a potent determinant that promotes transformation of mammary gland epithelial cells.(A) 67 deubiquitinase genes carried by retroviral vector were utilized to establish stably expression cell line based on mammary gland epithelial cell MCF10A, respectively. The individual 67 stable cell lines were subjected to soft agar colony formation assay, respectively. The capacity of each deubiquitinase in promoting the transformation of mammary gland epithelial cell was systematically evaluated. Several deubiquitinases were observed to promote colony formation. USP11 turns to be the most potent one that promotes the transformation of mammary gland epithelial cells. (B) Confirmation of USP11 in transformation of mammary gland epithelial cells. Wild-type USP11 and USP11 deubiquitinase catalytic death mutant (cysteine 318 is replaced by alanine) were stably delivered into MCF10 cells. While USP11-WT dramatically drove MCF10A cell for colony formation, USP11-C318A failed to transform MCF10A cells. (C) Acini overgrowth analysis for USP11. Both USP11-WT and USP11-C318A were subjected to an acini overgrowth assay. The count of colony size and number were shown in right panel. USP11-WT but not USP11-C318A mutant in promoting MCF10A acini overgrowth was observed. (D) Staining of active caspase 3 and DAPI in MCF10A acini culture. USP11-WT but not USP11-C318A mutant inhibits the activation of caspase 3 in central cell population of acini culture.