Fig 2: USP5 regulates aerobic glycolysis in TNBC. a–d Glucose update (a), lactate level (b), pyruvate level (c), and ATP level (d) were significantly decreased after USP5 knockdown in MDA-MB-231 cells. e, f ECAR values (e) and calculated glycolytic capacity (f) of control and USP5 knockdown MDA-MB-231 cells

Fig 3: USP5 promotes TNBC progression by regulating PFKP protein expression in vitro and in vivo. a The USP5 and PFKP protein expression in control and USP5 knockdown MDA-MB-231 cells in the presence or absence of overexpressed PFKP were analyzed by Western blot. b–f MDA-MB-231 cell proliferation following USP5 knockdown in the presence or absence of overexpressed PFKP was evaluated using CCK8 assay (b), Clone formation assay (c, d), and EDU incorporation assay (e, f). ***P < 0.001, **P < 0.01. g Representative images of the xenograft tumors in each group. h, i Tumor volume (h) and tumor weight (i) of nude mice in each group. **P < 0.01, *P < 0.05. j The PFKP protein expression in each sample of mice tumor tissue was analyzed by IHC staining. Scale bars, 100 μm

Fig 4: USP5 is a bona fide deubiquitinase that targets PFKP protein for deubiquitination and stabilization in TNBC. a Co-IP analysis of PFKP protein ubiquitination in MCF-10A and MDA-MB-231 cells. b MCF-10A and MDA-MB-231 cells were treated with CHX for the indicated time points and analyzed by Western blot. c Quantification of PFKP protein levels normalized to β-actin in time after the addition of CHX. d The diagram showing three deubiquitinases (USP5, USP14, and OTUB1) and three E3 ubiquitin ligases (HUWE1, TRIM21, and TRIM25) could interact with PFKP identified by a combination of Co-IP and MS. e The correlation between USP5 protein expression and PFKP protein expression in TNBC was analyzed by LinkedOmics platform. f The USP5 and PFKP protein expression in control and USP5 knockdown MDA-MB-231 cells were analyzed by Western blot. g Co-IP analysis of PFKP protein ubiquitination in control and USP5 knockdown MDA-MB-231 cells. h–k Control and USP5 knockdown MDA-MB-231 cells were treated with CHX for the indicated time points and analyzed by Western blot h, j, quantification of PFKP protein levels normalized to β-actin in time after the addition of CHX (i, k). l The binding of USP5 to PFKP was detected by Co-IP assay in MDA-MB-231 cells. m Immunofluorescence co-localization of USP5 and PFKP in MDA-MB-231 cells. n In vitro binding assays of purified GST-PFKP and His-USP5 proteins