Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Boxplot of the UCH-L1 mRNA expression levels in four different subtypes of BRCA from the GEPIA2 database. B mRNA expression levels of UCH-L1 in TNBC and normal breast tissues from the GEPIA2 database. C IHC analysis of TNBC ( n = 40) and normal breast tissue samples ( n = 20) was performed using anti-UCH-L1 antibody. Representative images of IHC staining and quantified data presented in box plots are shown. Scale bar, 100 μm. D Western blot analysis of UCH-L1 protein expression level in normal breast cell and multiple TNBC cell lines. β-Actin was used as a loading control. E Kaplan-Meier plots for the overall survival of TNBC patients according to UCH-L1 mRNA expression from the Kaplan–Meier Plotter database. F Kaplan-Meier plots for the relapse-free survival of TNBC patients according to UCH-L1 protein expression from the Kaplan–Meier Plotter database. G Kaplan-Meier plots for the overall survival of chemotherapy-treated TNBC patients according to UCH-L1 mRNA expression from the Kaplan–Meier Plotter database. H The receiver operating characteristic (ROC) curve between UCH-L1 expression and therapeutic responses to taxane-containing NAC in breast cancer cohorts. I The correlation analysis between the efficacy of taxane-based chemotherapy and UCH-L1 expression. Chi-square test was performed to calculate statistical significance between two comparator groups. Stacked bar chart showed the proportion of patients with or without response in high or low UCH-L1 groups. * p < 0.05; *** p < 0.001.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Expressing, Immunohistochemistry, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Knockdown efficiency of UCH-L1 in TNBC cells was evaluated by Western blot assay. B HCC1806 and BT549 cells with or without UCH-L1 depletion were incubated with indicated concentrations of PTX for 72 h, and cell viability was measured by CCK-8 assay. C Colony formation of HCC1806 and BT549 cells (500 cells per well) with or without UCH-L1 depletion in the presence of DMSO or 20 nM PTX. D HCC1806 and BT549 cells with or without UCH-L1 depletion were incubated with 20 nM PTX for 24 h, and cell proliferation was measured by EdU assay. Scale bars, 150μm. E HCC1806 and BT549 cells with or without UCH-L1 depletion were incubated with 20 nM PTX for 48 h, and cell apoptosis was measured by TUNEL assay. Scale bars, 150μm. F A schematic view of the animal study plan ( n = 6). G Curves of tumor growth. H Photopraphs of mice tumors of each group at the termination of the experiment. I Tumor weights were measured at the end of the experiment. J Characterization of HCC1806 xenograft tumors with histologic analysis by IHC staining of Ki67 and TUNEL. K The effect of treatment on mice body weight. Data are represented as mean ± SD of biological triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Knockdown, Western Blot, Incubation, CCK-8 Assay, EdU Assay, TUNEL Assay, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Volcano plot of quantitative proteomics showed the differentially expressed genes between UCH-L1 knockdown and control HCC1806 cells. B KEGG pathway analysis for differentially expressed proteins in HCC1806 cells with or without UCH-L1 knockdown. C Effect of UCH-L1 knockdown on the ECAR of HCC1806 cells was detected using the Seahorse analyzer. D Quantification analysis of glycolysis, glycolytic capacity and glycolytic reserve levels in the ECAR assays. E - G Glucose consumption, lactate and ATP production were determined in control and UCH-L1 knockdown HCC1806 and BT549 cells. H - J Glucose consumption, lactate and ATP production were determined in MDA-MB-231 cells with or without UCH-L1 overexpression. K - L Colony formation of MDA-MB-231 cells with or without UCH-L1 overexpression in the presence of DMSO or PTX and 2-DG. M MDA-MB-231 cells from the indicated groups were incubated with PTX for 72 h, and cell viability was measured by CCK-8 assay. N MDA-MB-231 cells stably expressing empty vector (EV) or flag-tagged UCH-L1 were incubated with DMSO or PTX and 2-DG for 24 h, and cell proliferation was measured using EdU. Scale bars, 150 μm. O MDA-MB-231 cells with or without UCH-L1 overexpression were incubated with DMSO or PTX and 2-DG, and cell apoptosis was measured using TUNEL assay. Scale bars, 100 μm. Results shown are representative of three independent experiments. Data are represented as mean ± SD of biological triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Quantitative Proteomics, Knockdown, Control, Over Expression, Incubation, CCK-8 Assay, Stable Transfection, Expressing, Plasmid Preparation, TUNEL Assay

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A The top twenty UCH-L1-interacting proteins according to the number of identified unique peptides by Co-IP-MS assay. B HEK293T cells were transfected with Flag-PKM2 and Myc-UCH-L1 plasmids, and then subjected to immunoprecipitation with anti-Flag or anti-Myc antibodies. The lysates and immunoprecipitates were then blotted. C Immunoprecipitation analysis with the indicated antibodies was performed to detect endogenous UCH-L1 and PKM2 interaction in BT549 cells. D Immunofluorescence assay to confirm the colocalization of UCH-L1 and PKM2 in HCC1806 cells. E Purified recombinant GST-UCH-L1 interacts with PKM2. GST-UCH-L1 and GST proteins were pulled down with glutathione beads. PKM2 was detected by Western blot. F Schematic representation of full-length (FL) UCH-L1, PKM2 and different truncation mutants. G Myc-UCH-L1 FL or indicated truncation mutants were co-expressed with Flag-PKM2 in HEK293T cells. Flag-PKM2 was immunoprecipitated with anti-Flag antibody, followed by immunoblotting analysis of Myc-UCH-L1 using Myc antibody. H Flag-PKM2 FL or indicated truncation mutants were co-expressed with Myc-UCH-L1 in HEK293T cells. Myc-UCH-L1 was immunoprecipitated with anti-Myc antibody, followed by immunoblotting analysis of Flag-PKM2 using Flag antibody. I MDA-MB-231 cells transfected with the indicated plasmid were incubated with PTX for 72 h, and cell viability was measured by CCK-8 assay. J , K MDA-MB-231 cells with UCH-L1 overexpression were transfected with PKM2 siRNA, and cell viability was detected by CCK-8 assay after 72 h incubation of the indicated concentrations of PTX. Results shown are representative of three independent experiments. Data are represented as mean ± SD of biological triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Immunofluorescence, Purification, Recombinant, Western Blot, Plasmid Preparation, Incubation, CCK-8 Assay, Over Expression

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Immunoblotting analysis of PKM2 and UCH-L1 in UCH-L1 knockdown TNBC cells. B Immunoblotting analysis of PKM2 and UCH-L1 in MDA-MB-231 and HEK293T cells transfected with increasing amounts of UCH-L1 WT or C90S. C HCC1806 cells with UCH-L1 depletion were transfected with UCH-L1 WT or C90S, and PKM2 expression was detected. D Immunoblotting analysis of PKM2 and UCH-L1 in UCH-L1 knockdown TNBC cells treated with or without MG132. E MDA-MB-231 cells transfected with UCH-L1 WT or C90S were treated with CHX for the indicated time. F Immunoblotting analysis of PKM2 and UCH-L1 in UCH-L1 knockdown TNBC cells treated with or without CHX for the indicated time. PKM2 expression was detected by Western blot. Quantitation of PKM2 protein level based on band intensity was shown. Results shown are representative of three independent experiments.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Western Blot, Knockdown, Transfection, Expressing, Quantitation Assay

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Flag-PKM2 and HA-ubiquitin (HA-Ub) were co-expressed with Myc-UCH-L1 WT or C90S in HEK293T cells. After MG132 treatment, IP was performed with Flag antibody, followed by immunoblotting with the indicated antibodies. B HA-Ub was co-expressed with Myc-UCH-L1 WT or C90S in MDA-MB-231 cells. After MG132 treatment, IP was performed with PKM2 antibody, followed by immunoblotting with indicated antibodies. C HCC1806 cells with or without UCH-L1 depletion were transfected with HA-Ub. IP was performed with PKM2 antibody after MG132 treatment, followed by immunoblotting with indicated antibodies. D Ubiquitinated PKM2 was purified from HEK293T cells transfected with HA-Ub, and then incubated with GST-tagged wild-type UCH-L1, and UCH-L1 C90S proteins in reaction buffer. The reactions were terminated by boiling in 1X SDS sample buffer and analyzed using Western blot with anti-HA antibody. E HA-WT, K6, K11, K27, K29, K33, K48, or K63 Ub were co-transfected with Flag-PKM2 and Myc-UCH-L1 into HEK293T cells. Cell lysates were collected after MG132 treatment for 8 h and subjected to ubiquitination assay, and the ubiquitination level of PKM2 was detected by HA antibody. F Flag-PKM2 (WT or indicated KR mutants) and HA-Ub were co-expressed in 293T cells with or without UCH-L1 knockdown. IP was performed with Flag antibody after MG132 treatment for 8 h, followed by immunoblotting with anti-HA. G HCC1806 cells expressing Flag-PKM2 WT or indicated KR mutants were transfected with UCH-L1 siRNA, followed by immunoblotting with indicated antibodies. H HCC1806 cells expressing indicated KR mutants of Flag-PKM2 were transfected with UCH-L1 siRNA, and then treated with CHX for the indicated time. Cell lysates were collected and subjected to immunoblotting analysis. Quantitation of Flag-PKM2 protein level based on band intensity was shown. Results shown are representative of three independent experiments.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Purification, Incubation, Knockdown, Expressing, Quantitation Assay

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Dose–response curves of HCC1806 and HCC1806/TaxR cells. The IC50 was determined according to a dose vs. response curve by GraphPad Prism. B Immunoblotting analysis of PKM2 and UCH-L1 in HCC1806 and HCC1806/TaxR cells. C The glucose consumption, lactate and ATP production were determined and compared in HCC1806 and HCC1806/TaxR cells. D HCC1806/TaxR cells from the indicated groups were incubated with PTX for 72 h, and cell viability was measured by CCK-8 assay. E Cell apoptosis of HCC1806/TaxR cells with indicated treatment was measured by TUNEL assay. Scale bars, 150 μm. F HCC1806/TaxR cells with or without UCH-L1 depletion were incubated with 20 nM PTX for 24 h, and cell proliferation was measured by EdU assay. Scale bars, 150 μm. G Colony formation of HCC1806/TaxR cells (300 cells per well) with or without UCH-L1 depletion in the presence of DMSO or 20 nM PTX. HCC1806/TaxR cells were subcutaneously injected into 6-week-old female nude mice and subjected to PTX treatment as planned ( n = 5). Tumor sizes were measured and calculated as curves shown ( H ). The mice tumors of each group were photographed at the termination of the experiment (I). Tumor weights were measured at the end of the experiment ( J ). Characterization of HCC1806/TaxR xenograft tumors with histologic analysis by IHC staining of Ki67 and TUNEL ( K ). Data are represented as mean ± SD of biological triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Western Blot, Incubation, CCK-8 Assay, TUNEL Assay, EdU Assay, Injection, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Deubiquitinase UCH-L1 confers paclitaxel resistance via stabilizing PKM2 to promote glycolysis in triple-negative breast cancer

doi: 10.1038/s41419-026-08521-7

Figure Lengend Snippet: A Boxplot of the PKM2 mRNA expression levels in TNBC and normal breast tissues from the GEPIA2 database. B Kaplan-Meier plots for the overall survival of chemotherapy-treated TNBC patients according to PKM2 mRNA expression from the Kaplan–Meier Plotter database. C The PKM2 expressions in non-responder and responder groups in breast cancer cohorts from ROC plotter server. The clinical response was evaluated based on pathological response. D Representative images of IHC of UCH-L1 and PKM2 in 40 TNBC tissues. The correlation analysis between UCH-L1 and PKM2 expression was performed using Fisher’s Exact Test. E Quantitative expression of IHC staining of PKM2 in high or low UCH-L1 groups. F Quantitative expression of IHC staining of UCH-L1 in high or low PKM2 groups. G The positive correlation between UCH-L1 and PKM2. The Pearson correlation test was used. H Mechanistic diagram of UCH-L1-catalyzed PKM2 deubiquitination in the regulation of glycolysis and chemoresistance. Depletion of UCH-L1 led to PKM2 degradation and downregulation, thereby inhibiting glycolysis and re-sensitizing TaxR-TNBC cells to chemotherapy.

Article Snippet: Antibodies used in immunoblotting: UCH-L1 (66230-1-Ig, 1:1000), PKM2 (60268-1-Ig, 1:1000), β-actin (66009-1-Ig, 1:5000), Myc (60003-2-Ig, 1:5000), HA (66006-2-Ig, 1:5000), Flag (66008-4-lg, 1:5000), GST (10000-0-AP, 1:4000) were purchased from Proteintech.

Techniques: Expressing, Immunohistochemistry