Fig 2: TDRD3 regulates USP9X localization to SGs in response to arsenite treatment. (a) HeLa cells cultured on glass coverslips were left untreated (Control) or treated with 0.5 mm sodium arsenite (Arsenite) for 30 min. The cells were fixed and immunostained with anti-USP9X and anti-TDRD3 to detect the localization of endogenous proteins. DAPI was used to stain the nuclear DNA. The arrows indicate USP9X and TDRD3 co-localization in SGs. (b) HeLa cells were treated as in a and immunostained with anti-USP9X and anti-G3BP. (c) Wild-type and TDRD3-knockout MEF cells were both treated with 0.5 mm sodium arsenite for 30 min. The cells were fixed and immunostained with anti-USP9X and anti-G3BP antibodies.

Fig 3: Aortic valve disease in mice with Usp9x deletion in endothelial cell lineage.(A) Breeding scheme to generate mice harboring endothelial and endothelial-derived cell deletion of Usp9x (Usp9xfl/fl; Tie2Cre). (B) Experimental scheme for Usp9xfl/Y; Cre+ males and Usp9xfl/Y; Cre- controls. (C) Survival in Usp9xfl/Y; Cre+ male mice at P10. (D) Aortic velocity measured by echocardiogram in Cre+ males at 6, 16, and 24 weeks of age compared to Cre- controls. (E) Doppler image shows aortic valve regurgitation in Cre+ male at 24 weeks of age as compared to Cre- control. Table shows incidence of aortic valve regurgitation in Cre+ and Cre- males. (F) Histological section of aortic valve stained with Russell-Movat pentachrome shows increased proteoglycan deposition in Cre+ males compared to Cre- controls. (G) Graph shows quantification of aortic valve area in Cre+ and Cre- males. (H) Alizarin red staining demonstrates calcification in Cre+ males. (I) Table shows incidence of calcification in Cre+ and Cre- males. Scale bars, 100 µm (pentachrome staining) and 20 µm (Alizarin red staining). *P = 0.05 (two-tailed Mann-Whitney test).

Fig 4: USP9X and TTK knockdown partially suppres cell proliferation and NSCLC. (A) A549 cells stably expressing shGFP, shUSP9X or shTTK were subjected to a cell proliferation assay. Each point represents the mean ± s.d. for biological quintuplicate experiments (n = 5, *** P < 0.001, two-way ANOVA). (B) Tumor images of nude mice (BALB/c; Slac Laboratory Animal, Shanghai, China) with subcutaneous injection of 5×106 A549 cells stably transfected with shGFP, shUSP9X or shTTK at day 28 after implantation. (C) Weights of tumors in (B) were measured. Each bar represents the mean ± s.d. for different mice groups (n = 6, *** P < 0.001, one-way ANOVA). (D) Tumor volumes were measured weekly. Each bar represents the mean ± s.d. for different mice groups (n = 6, *** P < 0.001, two-way ANOVA).

Fig 5: A schematic diagram of proposed crosstalk among acetylation, phosphorylation, and ubiquitination events in dynamic regulation of MCL1 protein stabilityMCL1 is an unstable protein that is targeted for ubiquitination. p300-directed MCL1 acetylation at K40 leads to enhanced interaction with USP9X, facilitating MCL1 deubiquitination and stabilization. SIRT3 is the potential deacetylase counteracting the acetylation-dependent MCL1 stabilization. Phosphorylation also plays important roles in regulating the interaction with USP9X and certain E3s. Elevated MCL1 acetylation status may correlate with sensitivity to USP9X and p300 inhibitors.