Fig 2: Identification of USP9X interactors in human dermal fibroblast cell lines derived from control individuals and individuals with USP9X mutationsThe USP9X mutations (p.Ile79Val, p.Ala696Val and p.Ser2233Pro) are known to cause neurodevelopmental disorders and predicted to disrupt binding of USP9X to different sub-sets of interacting proteins depending on the location of the mutation.(A) Western blot analysis of 3 replicate USP9X IP experiments. IgG IPs served as negative control samples in subsequent proteomics. USP9X and IgG control immunoprecipitated samples from each experiment subsequently analyzed using TMT LC-MS/MS identification and quantification.(B) Volcano plot of proteins immunoprecipitated with USP9X from control fibroblasts. Results derived from 3 independent IP experiments in (A). Fold change and statistical values represents comparison to proteins immunoprecipitated with control IgG. p values derived from adjusted Students paired t-test.(C) Relative protein quantities of significantly enriched USP9X interactors (enriched in USP9X IPs compared to IgG IPs in control cells) in mutant USP9X IP experiments. ∗p<0.05 paired Student’s t-test. Data is represented as mean ± standard deviation. Data is reproduced from Johnson et al. (2020) with permission from Elsevier.

Fig 3: CDC14B and CDK1 regulate phosphorylation of USP9X at serine 2563 in mitosis.a Co-immunoprecipitation of FLAG-tagged CDC14B with endogenous USP9X from HEK 293T cells that were either left untreated or arrested in mitosis using nocodazole (EV = expression vector). Immunocomplexes and respective WCE were probed with antibodies to the indicated proteins. b Immunoblot analysis of U2OS cells that were either synchronized in G1/S phase using a double thymidine block (left panel), or in mitosis using sequential thymidine and nocodazole treatment (right panel). Cells were then released into the cell cycle and collected at the indicated time points. c Immunoblot analysis of U2OS cells that were treated with siRNA directed against CDC14B and synchronized in mitosis as described above. Mitotic shake-off was performed and samples were analyzed by western blot with the indicated antibodies. d Quantification of n = 3 biologically independent experiments conducted as described in c. Ratio paired t-test was applied with **p = 0.0045. e Immunoblot analysis of mitotic U2OS cells after synchronization with thymidine and nocodazole. Mitotic cells were shaken off and kept in nocodazole-containing medium during treatment with either the CDK1 inhibitor RO-3306 or DMSO for 0.5 h. f In vitro kinase assay with C-terminal truncates of USP9X (aa 2165–2570), either the USP9X wild-type form (USP9XWT) or a USP9X form with a serine to alanine mutation on position 2563 (USP9XS2563A), that were purified from Escherichia coli and exposed to recombinant active CDK1-Cyclin B in the presence of radioactive 32P-ATP (CBB, Coomassie Brilliant Blue), *Cyclin B. g Quantification of two independent experiments conducted as described in f. 32P signals are normalized to the respective Coomassie signal. Mean is displayed from n = 2 biologically independent experiments. h Enzyme kinetics of USP9XWT and USP9XS2563A proteins purified from mitotic HEK 293T cells were measured at different Ubiquitin-AMC concentrations. The resulting values for KM of USP9XWT or USP9XS2563A were 0.9090 or 0.7835 µM, respectively, and for Vmax 36.69 or 31.06 µM/min, respectively. The values indicated are from n = 1 experiment. Throughout this figure means and standard deviations as error bars are displayed.

Fig 4: The ZBTB38 protein accumulates in H2O2-treated cells, and the accumulation depends on ROS and USP9X activity. (A) Western blot analysis in HeLa cells exposed to H2O2 at the indicated concentrations for 30 min. P-H2AX: phosphorylated H2AX. Hydroxyurea treatment (HU) was used as a positive condition for H2AX phosphorylation. (B) ZBTB38 mRNA levels in samples from panel A. qRT-PCR normalized with three housekeeping genes (MAPK14, TBP and TFRC), n = 3. (C) Left panel: treatment of HeLa cells with Etoposide (Etop.; 200 μM for 1 h) or Hydroxyurea (HU; 2 mM for 2 h) causes DNA damage, but no ZBTB38 accumulation. Right panel: Etoposide or Hydroxyurea do not increase the intracellular ROS levels, as measured by DHE staining. The mean DHE intensity is indicated on the graph. (D) Extracellular Superoxide Dismutase (SOD) or Catalase (Cat.) do not prevent ZBTB38 accumulation in response to H2O2 treatment (200 μM for 30 min), but PEG-coupled SOD or Cat., which are cell-permeable, do prevent ZBTB38 accumulation. (E) USP9X, but not USP7, accumulates in cells treated with H2O2 (200 μM for 30 min). (F) ZBTB38 protein does not accumulate in response to H2O2 in cells depleted of UPS9x. (G) Inhibition of USP9X by WP1130 prevents ZBTB38 accumulation in response to H2O2 treatment.

Fig 5: Usp9x suppresses CRC.(A) Schematic of AOM/DSS-induced CRC model. (B) H&E on sections from transformed colons of the indicated genotypes. Arrowheads indicate tumors. Scale bars: 100 mm. Right panels: ×4 magnification of the dashed areas on the left; dashed lines outline tumors. (C) Number of tumors in each mouse from experiment in B; n = 5–13/group. (D) Tumor area represented as mm2 in mice from B; n = 5–13/group. (E) Western blots showing levels of indicated proteins in 4 independent tumors from B. (F) H&E and c-Myc staining on sections from transformed colons of the indicated genotypes. Scale bars: 50 mm (H&E); 100 μm (c-Myc). Arrowheads indicate tumors. (G) Average number of tumors in indicated mice from experiment in F; n = 3–5/group. Data are presented as mean; statistical significance calculated by 1-way ANOVA in C and D, and by Student’s t test in G.