Fig 2: 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 3: 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

Fig 4: USP5 is positively correlated with PFKP expression in TNBC and is associated with poor prognosis of TNBC patients. a Representative images of IHC staining of USP5 and PFKP in serial sections of the same TNBC tumor. Scale bars, 100 μm. b A positive correlation between the expression of USP5 and PFKP in the 172 human TNBC carcinoma samples. r = 0.223, P = 0.003. c Concomitantly high expression of USP5 and PFKP correlates positively with the TNM stage in human TNBC carcinoma samples. r = 0.369, P < 0.001. d Kaplan–Meier curves showed that the overall survival of patients with concomitantly high USP5 and PFKP expression in TNBC tumors was shorter than that of those with concomitantly low USP5 and PFKP expression. Log-rank P = 0.0004. e A schematic diagram of USP5 promotes TNBC progression and aerobic glycolysis through deubiquitinating and stabilizing PFKP

Fig 5: Inhibition of PFKP reduces TNBC progression in vitro and in vivo. a, b qRT-PCR (a) and western blot (b) analysis of PFKP expression with PFKP knockdown in MDA-MB-231 cells. ***P < 0.001. c CCK-8 assay determination of MDA-MB-231 cell proliferation in response to PFKP knockdown. ***P < 0.001. d, e Clone formation assay determination of MDA-MB-231 cell proliferation in response to PFKP knockdown. ***P < 0.001, **P < 0.01. f, g EDU incorporation assay determination of MDA-MB-231 cell proliferation in response to PFKP knockdown. **P < 0.01. h Representative images of the xenograft tumors in each group. i, j PFKP knockdown reduces tumor volume (i) and tumor weight (j) of nude mice. ***P < 0.001. k The PFKP protein expression in each sample of mice tumor tissue was analyzed by IHC staining. Scale bars, 100 μm