Fig 1: Identification of USP13 as a candidate MCL1 deubiquitinase. a The quantification of MCL1 protein levels upon knockdown of each DUB in HEK293T cells. 84 siRNA pools were individually transfected into HEK293T cells, endogenous MCL1 was detected by western blotting and protein bands were quantified using the Image J software. b Classification of the 84 human DUBs included in siRNA screen. c Dot plot of copy number alterations for 20 candidate MCL DUB genes in TCGA cancers. d Validation of the candidate MCL1 DUBs in HEK293T cells. Four different siRNAs targeting USP9X, OTUB2, or USP13 were transfected into HEK293T cells, and endogenous expression levels of indicated proteins were measured by western blot analysis
Fig 2: OTUB2 promotes malignant progression of TNBC cells through EIF4A3/TPI1 mediated glycolysis. The infected or co-infected MDA-MB-468 cells were grouped as follows: sh-NC, sh-OTUB2, sh-OTUB2 + oe-EIF4A3, and sh-OTUB2 + oe-EIF4A3 + sh-TPI1. A Western blot detection of TPI1 protein. **p < 0.01; ***p < 0.001. B Glucose uptake, C ATP level, and D lactate production was measured by corresponding kits. *p < 0.05; **p < 0.01; ***p < 0.001. E The ECAR of MDA-MB-468 cells was analyzed using ECAR assay. F CCK-8 and G Transwell assays were used to evaluate cell proliferation, migration, and invasion. *p < 0.05; **p < 0.01; ***p < 0.001. n = 3 per group; Results are expressed as mean ± SD. Statistical analyses were analyzed using an one-way ANOVA followed by Tukey’s multiple-comparisons test
Fig 3: OTUB2 regulates EIF4A3 to promote malignant progression of TNBC cells. MDA-MB-468 cells were infected with sh-OTUB2 or co-infected with sh-OTUB2 and oe-EIF4A3. A and B qPCR and western blot were used to detect the mRNA and protein expressions of OTUB2 and EIF4A3. *p < 0.05; **p < 0.01; ***p < 0.001. C CCK-8, D colony-forming, and E Transwell assays were performed to evaluate cell proliferation, migration and invasion. **p < 0.01; ***p < 0.001. F Glucose uptake, G ATP level, and H lactate production in MDA-MB-468 cells were measured by corresponding kits. *p < 0.05; **p < 0.01. I The ECAR of cells was analyzed using ECAR assay. n = 3 per group; Results are expressed as mean ± SD. Statistical analyses were analyzed using one-way ANOVA followed by Tukey’s multiple-comparisons test
Fig 4: OTUB2 stabilizes EIF4A3 by deubiquitination. A TCGA database was used to analyze EIF4A3 expression in breast cancer tissues and TNBC tissues. ***p < 0.001. B Western blot detected the expression of EIF4A3 in TNBC tissues and para-carcinoma tissues (n = 30 samples per group). **p < 0.01. C Western blot detected the EIF4A3 protein in MCF10A, MDA-MB-231, MDA-MB-453, MDA-MB-468, SUM-159, and BT-549 cells. *p < 0.05; **p < 0.01. D and E MDA-MB-468 cells were infected with sh-OTUB2, sh-NC, oe-OTUB2, and oe-NC, respectively. Relative mRNA and protein expressions of OTUB2 and EIF4A3 were detected. **p < 0.01; ***p < 0.001. F Co-IP assay for measurement of interaction between OTUB2 and EIF4A3. OTUB2 and EIF4A3 protein expressions were evaluated in anti-OTUB2 complex or anti-IgG by western blot. G MDA-MB-468 cells infection with sh-OTUB2 or sh-NC were treated with CHX for 0, 2, 4, 6, 8 h, respectively. EIF4A3 protein was measured by western blot. *p < 0.05; **p < 0.01. H MDA-MB-468 cells infection with sh-OTUB2 or oe-OTUB2 were treated with MG132 for 6 h. Ubiquitination assay was used to test the effect of OTUB2 on EIF4A3 ubiquitination. I In vitro ubiquitination assay. Purified V5-ubiquitin and Flag-EIF4A3 were incubated with either HA-OTUB2 or HA-OTUB2C51S. Then, Flag-U2AF2 proteins were further purified with anti-Flag antibody and immunoblotted with antibodies against V5 and Flag. n = 3 per group; Results are expressed as mean ± SD. Statistical analyses were analyzed using a Student’s t-test in A (left) and B, while one-way ANOVA followed by Tukey’s multiple-comparisons test were used to analyze differences between groups in A(right), C-E, and G
Fig 5: OTUB2 facilitates TNBC malignant progression via EIF4A3/TPI1 in vivo. MDA-MB-468 infected with sh-OTUB2 or sh-NC, and BT-549 cells infected with oe-OTUB2 or oe-NC were subcutaneously injected into the right thoracic mammary fat pads of nude mice, respectively (n = 5 mice per group). A Tumor images. B Tumor volume was measured every 7 days. *p < 0.05;***p < 0.001. C Tumor weight was recorded after 35 days. *p < 0.05; **p < 0.01. D The protein expressions of OTUB2, EIF4A3, and TPI1 were detected. E The Ki-67, OTUB2, EIF4A3, and TPI1 proteins were evaluated using immumohistochemical assay. *p < 0.05; **p < 0.01; ***p < 0.001. Results are expressed as mean ± SD. Statistical analyses were analyzed using a Student’s t-test in B, while one-way ANOVA followed by Tukey’s multiple-comparisons test were used to analyze differences between groups in C and D
Supplier Page from Abcam for Anti-OTUB2 antibody