Fig 2: Evaluation of apoptosis regulating proteins after lexatumumab (1 µg/ml) and DTIC (50 µg/ml) treatment in FEMX-1 and HHMS.The cells were harvested 24 h after treatment exposure and analyzed for various apoptotic markers. (A) Immuno-blot detection of Bcl-w, Bcl-XL, Mcl-1, Bak, Bax, Bik, Bad, BimEL, BimL and BimS expression in FEMX-1 cells. (B) Immuno-blot detection of cIAP-1, cIAP-2, XIAP, survivin and livin in FEMX-1 and HHMS cells. ß-actin is used as loading control. Cell lysates were prepared from three independent biological experiments, and representative blots are included in the figure.

Fig 3: Molecular characterizations of MCF7 and MCF7-TAM12.5 cells. (A) Protein expression level of ERa, ERß, HER2, p53, MDR1, Smac, XIAP, and Bcl-2 was analyzed in MCF7, MCF7-TAM12.5 (under 12.5 µM tamoxifen), and MCF7-TAM12.5 (drug free) cells by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are the intensities of the blot relative to MCF7 cells. (B) Location of ERa and HER2 (green fluorescence) was visualized by immunofluorescence microscopy. Nuclei were counterstained blue by DAPI.

Fig 4: USP9X binds, deubiquitylates, and stabilizes XIAP in mitosis Immunoblot analysis of HeLa cells using the indicated antibodies that were synchronized in G1/S phase using a double thymidine block or in mitosis using sequential thymidine and nocodazole treatment and collected at the given time points.Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides as indicated and left untreated or synchronized in mitosis using sequential thymidine and nocodazole treatment.Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were transfected with FLAG-tagged XIAP or empty vector (EV) and synchronized in mitosis using nocodazole or left untreated.Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were treated as in (C), but with additional transfection of the FLAG-tagged XIAP single amino acid mutants G188R and G188E. Only mitotic samples are shown. The asterisk denotes ubiquitylated forms of XIAP. In vivo ubiquitylation of XIAP in HeLa cells that were infected with the indicated expression constructs carrying FLAG-tagged XIAP and transfected with siRNA oligonucleotides as specified. Cells were synchronized in mitosis using sequential thymidine/nocodazole treatment, as indicated. Subsequent to treatment with MG132, whole-cell extracts (WCE) were prepared and ubiquitylated XIAP was isolated by anti-FLAG immunoprecipitation (IP) under denaturing conditions.Immunoblot analysis of NIH 3T3 cells that were transfected with expression constructs for USP9XWT or the catalytically inactive mutant USP9XC1556S. The band in the EV control lane of the anti-V5 panel marks an unspecific band produced by the antibody.Immunoblot analysis of HeLa cells using antibodies to the indicated endogenous proteins that were synchronized in mitosis using thymidine/nocodazole and treated with DMSO or the USP9X inhibitor WP1130 as indicated. Source data are available online for this figure.

Fig 5: High XIAP mRNA expression level is positively correlated with poor survival of breast cancer patients. (A) The mRNA expression levels of XIAP in normal and tumor tissues of patients with estrogen receptor–negative and estrogen receptor–positive breast cancer were analyzed using the database and software available online (OncomineTM) (see text footnote 1). (B) Kaplan–Meier survival estimates of high (red line) or low (black line) XIAP expression in ER+ tamoxifen-treated luminal A breast cancer subtype. Analysis was performed using the online database and web tool (Kaplan–Meier plotter, https://kmplot.com/).