Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 1. Usp28 is expressed in intestinal crypts and controls intestinal differentiation and proliferation. (A and B) Expression and localization analysis of USP28 in murine intestine. (A) Usp28fl/fl mice. Boxed regions are shown at higher magnification at right. Bottom right: Usp28 was expressed in crypt base columnar cells (red arrowhead), whereas Paneth cells were devoid of USP28 staining (green arrow). Top right: Usp28 expression gradually decreased within the transit-amplifying cell compartment and was lost in the upper half of the crypt, which contains differentiated cells. (B) Usp28ΔG mice showed no USP28 staining in intestinal tissue. (C) Staining for GFP to label Lgr5+ stem cells in an Lgr5-GFP mouse. (D) Usp28 expression in crypt cells sorted for Lgr5-GFP. (E and F) Representative sections of villi (E) and quantification of goblet cells (AB/PAS+; F), showing increased numbers of goblet cells in Usp28ΔG intestines. n = 10 per group. (G) Representative crypt sections showing mislocalization of Paneth cells (black arrowheads) in Usp28ΔG animals. (H) Quantification of Paneth cells (lysozyme+) per crypt. n = 10 per group. (I and J) Usp28ΔG animals had fewer proliferating cells (BrdU pulse 2.5 hours) within the crypt. n = 10 per group. (K) qRT-PCR of RNA from isolated Usp28ΔG crypt cells showing decreased progenitor and increased differentiation marker gene expression. Expression was normalized to actin and is shown relative to control Usp28fl/fl cells (assigned as 1.0; dashed line). n = 5 per group. Original magnification, ×10 (E); ×20 (C, top); ×40 (A, left; B; C, bottom; G; and I); ×80 (A, right). (D, F, H, J, and K) ***P < 0.0001, *P < 0.001, Student’s t test. Error bars indicate SEM.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Expressing, Staining, Quantitative RT-PCR, Isolation, Marker, Gene Expression, Control

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 2. Usp28ΔG intestinal tissue shows decreased c-JUN and NICD1 as well as c-MYC protein. (A) Western blots of isolated crypt cell lysates from Usp28ΔG intestines show lower USP28, NICD1, phospho– and total c-MYC, and phospho– and total c-JUN proteins compared with Usp28fl/fl controls. (B) Quantification of A. (C) Levels of USP25 and phospho– and total β-catenin were unchanged in crypt cell lysates from Usp28ΔG intestines compared with Usp28fl/fl controls. USP28 and actin are shown as controls. (D) qRT-PCR of RNA from isolated Usp28ΔG crypt cells showing decreased expression of Usp28 (black bars) and c-JUN, NICD1, and c-MYC target genes (white bars) compared with Usp28fl/fl controls, whereas expression of Notch1 and Myc (gray bars) was unaltered. Dll1, which is negatively regulated by Notch signaling, is an example of mRNA increased in Usp28ΔG cells. Expression was normalized to actin and is shown relative to Usp28fl/fl control (assigned as 1.0; dashed line). n = 3 animals per genotype. Error bars indicate SEM.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Western Blot, Isolation, Quantitative RT-PCR, Expressing, Control

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 3. c-JUN and NICD1 are deubiquitinated and stabilized by USP28. (A) NICD1, c-MYC, cyclin E1, and c-JUN protein levels were decreased in HCT116 cells transiently transfected with 2 independent shRNA constructs against USP28 (sh-USP28-1 and sh-USP28-2) compared with nontargeting control shRNA (sh-scrambled). Vinculin was used as a loading control. Images are representative of 2 independent experiments. (B) Time course of cycloheximide (CHX) treatment of HCT116 cells transiently transfected with scrambled control shRNA or shRNA targeting USP28. Protein stability of USP28, NICD1, and c-MYC was analyzed at the indicated times by Western blot. CDK2 and vinculin were used as controls. Images are representative of 3 independent experiments. (C) Endogenous USP28 coimmunoprecipitated NICD1, c-MYC, and c-JUN. Images are representative of 3 independent experiments. (D) USP28 promoted deubiquitination of c-JUN and NICD1. HeLa cells transfected with c-JUN or NICD1 together with His-tagged ubiquitin, with or without overexpres- sion of USP28 or the catalytically impaired USP28 C171A, were treated 2 days after transfection with MG132 for 3 hours and lysed, after which protein com- plexes were immunoprecipitated using nickel-NTA beads. Ubiquitinated (Ubi-) complexes were analyzed by Western blot against c-JUN or NICD1. Images are representative of 3 independent experiments.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Transfection, shRNA, Construct, Control, Western Blot, Ubiquitin Proteomics, Immunoprecipitation

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 4. USP28 is commonly overexpressed in human colorectal carcinomas. (A) Heat map of APC, USP28, and c-MYC expression in human intestinal samples from tumors of mucosa and crypt epithelium (adenoma or carcinoma) and the corresponding normal tissue. Data were obtained from Oncomine and Skrzypczak public databases (see Methods). (B) Human colorectal tissue microarray samples from control sections or grade 1 tumor sections stained with antibodies against USP28, c-JUN, and c-MYC. Bottom row shows higher magnifications of the same samples. Original magnification, ×10 (top); ×40 (bottom). (C) Staining intensity was blind-assessed and quantified (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) in tumor sec- tions of grade 1–3 and control (C) tissue sections from the tissue microarray (total 80 samples). *P < 0.05.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Expressing, Microarray, Control, Staining

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 5. USP28 is a c-MYC target gene that is highly expressed in murine as well as human intestinal tumors. (A) H&E- and USP28-stained sections of the small intestine of an Apcmin/+ animal, including adenomas (dashed outlines). Red and green boxed regions denote tumor and control image areas, respectively. USP28 staining was higher in tumor tissue compared with control crypts and villi. Scale bars: 2.5 mm (gut roll); 100 μm (low-power views); 25 μm (high-power views). (B) qRT-PCR of RNA isolated from Apcmin/+ tumors showed increased Usp28 expression compared with WT tissue. n = 5 per group. Error bars indicate SEM. (C) Human USP28 locus, showing the c-MYC binding site (E-box) upstream of the transcription start site. Small arrows indicate primers used for ChIP in D. (D) ChIP from HCT116 cells. c-MYC efficiently bound to the E-box within the USP28 promoter, but not to an unrelated control locus (see Methods). Data are representative of 3 independent experiments. (E) qRT-PCR validation of c-MYC knockdown and effect on USP28 expression. Error bars indicate SEM. Data are representative of 2 independent experiments. (F) Western blot analysis of HCT116 cells 48 or 72 hours after siRNA transfection. Knockdown of c-MYC decreased USP28 protein levels.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Staining, Control, Quantitative RT-PCR, Isolation, Expressing, Binding Assay, Biomarker Discovery, Knockdown, Western Blot, Transfection

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 6. USP28 deficiency ameliorates tumorigenesis in Apcmin/+ animals. (A) Kaplan-Meier diagram showing increased lifespan of Apcmin/+ Usp28ΔG (n = 14) compared with Apcmin/+ Usp28fl/fl (n = 22) mice. (B) Average tumor size in the small intestine at 18 weeks of age was reduced in Apcmin/+ Usp28ΔG mice. n = 5 per group. (C) Number of tumors in the small intestine and colon at 18 weeks of age is reduced in Apcmin/+ Usp28ΔG mice. n = 5 per group. (D) Quantification of BrdU-incorporating cells per mm2 area of tumor section showing decreased proliferation in Apcmin/+ Usp28ΔG tumors. n = 4 per group. (E) Representative sections of tumors (black dashed outline) of the small intestine stained with H&E or AB/PAS to stain goblet cells. Boxed regions are shown at higher magnification. Quantification of goblet cells is also shown. (F) Sections of tumors (white dashed line) within the small intestine of 18 week-old Apcmin/+ Usp28fl/fl and Apcmin/+ Usp28ΔG animals, stained with H&E (left) and USP28 antibody (right, enlarged). Black dashed box indicates enlarged area. (G) Lysates from isolated small intestinal Apcmin/+ tumors show decreases in the indicated proteins in Usp28ΔG tumors when analyzed by western blot- ting. Actin was used as loading control. (H) qRT-PCR of RNA isolated from small intestinal tumors showing expression of genes positively (Hes1, Hes5) and negatively (Dll1) regulated by NICD1. n = 5 per group. Data represent means. Original magnification, ×5 (E, left and middle, and F, left); ×40 (E and F, right). (B–E and H) Where shown, error bars indicate SEM. *P < 0.05, **P < 0.005.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Staining, Isolation, Western Blot, Control, Quantitative RT-PCR, Expressing

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 7. Inducible Usp28 deletion reduces tumor burden and improves survival in Apcmin/+ animals. (A) Inducible Usp28 deletion. Cre expression was induced by 5 consecutive days of i.p. tamoxifen injection at 110 days of age, when intestinal tumors were already established. R26CreERt, Rosa26 Cre-ERT2. (B) Kaplan-Meier diagram showing survival of tamoxifen-injected Apcmin/+ Usp28fl/fl and Apcmin/+ Usp28fl/fl Rosa26 Cre-ERT2 mice. n = 5 per genotype. (C) Average tumor size in the small intestine at endpoint. n = 5 per group. (D) Quantification of Ki67+ cells per mm2 in tumors at endpoint. (E) AB/PAS+ goblet cells per mm2 in tumors at endpoint. (F) Number of tumors in the small intestines of mice from A postmortem. n = 5 per group. (G) Acute loss of Usp28 reduced cell proliferation in tumors. Representative immunohistological sections of tumors from tamoxifen-treated Apcmin/+ Usp28fl/fl and Apcmin/+ Usp28fl/fl Rosa26 Cre-ERT2 mice, 2 days after acute Usp28 deletion, stained for phospho–histone 3 (pH3). Scale bars: 50 μm. (H) pH3+ cells per mm2 in tumors 2 days after acute Usp28 deletion. (I) Apcmin/+ tumors stained with cleaved caspase 3, showing increased cell death after acute Usp28 deletion. Scale bars: 50 μm. (J) Cleaved caspase 3+ cells per mm2 in tumors 2 days after acute Usp28 deletion. (C–F, H, and J) Error bars indicate SEM. Symbols in D, E, H, and J indicate individual tumors. ***P < 0.001, *P < 0.05.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Expressing, Injection, Staining

Journal: Cell death & disease

Article Title: USP28 promotes PARP inhibitor resistance by enhancing SOX9-mediated DNA damage repair in ovarian cancer.

doi: 10.1038/s41419-025-07647-4

Figure Lengend Snippet: Fig. 2 Olaparib enhances the interaction between USP28 and SOX9. A GST pull-down assay was used to validate the direct interaction between SOX9 and USP28. B The schematic diagram illustrates the full-length and truncation mutants of USP28 proteins. C The schematic diagram illustrates the full-length and truncation mutants of SOX9 proteins. D Full-length and truncated HA-tagged USP28 were co-transfected into HEK293T cells along with Flag-SOX9. Co-IP assay was performed using anti-HA beads and co-eluted SOX9 was detected using anti-Flag antibody. E Full-length and truncated Flag-tagged SOX9 were co-transfected into HEK293T cells along with HA-USP28. Co-IP assay was performed using anti-Flag beads and co-eluted USP28 was detected using anti-HA antibody. F and G Co-IP was performed to verify the endogenous interaction between SOX9 and USP28. H Cells were treated with olaparib (Ola) for 24 h, followed by pulldown with anti-USP28 antibody and immunoblotting with the antibodies indicated. Quantification of the IPed SOX9 protein level was shown in Supplementary Fig. S2C. I Cells were treated with olaparib (Ola) for 24 h, followed by pulldown with anti-FBXW7 antibody and immunoblotting with the antibodies indicated. J Quantification of the IPed SOX9 protein level in (I). K Western blot was used to detect the SOX9 Co-IPed with anti-USP28 antibody in SKOV3 and SKOV3/Ola cells. L Quantification of the IPed SOX9 protein level in (K). M Western blot was used to detect the SOX9 Co-IPed with anti- FBXW7 antibody in SKOV3 and SKOV3/Ola cells. N Quantification of the IPed SOX9 protein level in (M). (Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, n = 3).

Article Snippet: Antibodies against GST (10000-0-AP), poly (ADP-ribose) polymerase 1 (PARP1, 13371-1-AP), His (66005-1-Ig), Flag (20543-1-AP), MYC (16286-1- AP), HA (81290-1-RR), SMARCA4 (21634-1-AP), USP25 (12199-1-AP), and USP28 (17707-1-AP) were procured from Proteintech (Wuhan, China).

Techniques: Pull Down Assay, Transfection, Co-Immunoprecipitation Assay, Western Blot

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP24 suppresses ferroptosis in triple-negative breast cancer by stabilizing DHODH protein

doi: 10.1038/s41419-025-07895-4

Figure Lengend Snippet: A Schematic of the anticancer effect of RSL3 (2 mg/kg/2 days) in the indicated MDA-MB-231 tumor xenograft models. B The tumor volume of each group was calculated every 2 days. C The excised tumors were photographed on day 22. D The tumor weights of each group. Mean ± SD, n = 6. * P < 0.05, *** P < 0.001, **** P < 0.0001. E The mice body weight of each group was recorded every 2 days. F The expression of USP24, DHODH, Ki67, 4-HNE, and cleaved-caspase 3 were detected by immunohistochemical staining. Representative images and quantification in each group were shown (n = 3 samples). Scale bar, 20 μm. Mean ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns no significance.

Article Snippet: USP5 (10473-1-AP), USP9X (81892-1-RR), USP10 (19374-1-AP), USP13 (16840-1-AP), USP24 (13126-1-AP), USP28 (17707-1-AP), USP38 (17767-1-AP), USP48 (12076-1-AP), USP53 (83846-1-RR), DHODH (14877-1-AP), GSS (15712-1-AP), ACSL4 (22401-1-AP), NRF2 (80593-1-AP), Ki67 (27309-1-AP), and FSP1 (20886-1-AP) antibodies were purchased from Proteintech (Chicago, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP24 suppresses ferroptosis in triple-negative breast cancer by stabilizing DHODH protein

doi: 10.1038/s41419-025-07895-4

Figure Lengend Snippet: A Schematic of the anticancer effect of RSL3 (2 mg/kg/2 days) with or without WP1130 (25 mg/kg/2 days) in the MDA-MB-231 tumor xenograft models. B The tumor volume of each group was calculated every 2 days. C The excised tumors were photographed on day 22. D The tumor weights of each group. Mean ± SD, n = 6. **** P < 0.0001. E The mice body weight of each group was recorded every 2 days. F The expression of USP24, DHODH, Ki67, 4-HNE, and cleaved-caspase 3 were detected by immunohistochemical staining. Representative images and quantification in each group were shown (n = 3 samples). Scale bar, 20 μm. Mean ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns no significance.

Article Snippet: USP5 (10473-1-AP), USP9X (81892-1-RR), USP10 (19374-1-AP), USP13 (16840-1-AP), USP24 (13126-1-AP), USP28 (17707-1-AP), USP38 (17767-1-AP), USP48 (12076-1-AP), USP53 (83846-1-RR), DHODH (14877-1-AP), GSS (15712-1-AP), ACSL4 (22401-1-AP), NRF2 (80593-1-AP), Ki67 (27309-1-AP), and FSP1 (20886-1-AP) antibodies were purchased from Proteintech (Chicago, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP24 suppresses ferroptosis in triple-negative breast cancer by stabilizing DHODH protein

doi: 10.1038/s41419-025-07895-4

Figure Lengend Snippet: A , B Cellular proteins in the indicated MDA-MB-231 and MDA-MB-468 cell lines was measured by Western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. C , D Western blotting analysis of DHODH proteins in MDA-MB-231 and MDA-MB-468 cells treated with the indicated doses of WP1130 for 24 h. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. ** P < 0.01, *** P < 0.001, **** P < 0.0001. E , F MDA-MB-231 cells were stably transfected with two individual USP9X shRNAs or control shRNA. USP9X expression in cells was measured by Western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. * P < 0.05, ** P < 0.01. ns no significance. G , H Western blotting analysis of cellular proteins in the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with cycloheximide (CHX, 100 μg/ml for MDA-MB-231 cells; 30 μg/ml for MDA-MB-468 cells) for the indicated time periods. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. ns no significance. I , J MDA-MB-231 cells were treated with MG132 (10 μM) or chloroquine (CQ, 30 μM) for the indicated time periods. The protein levels of DHODH were measured by Western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. * P < 0.05,, *** P < 0.001. ns no significance. K , L The indicated MDA-MB-231 and MDA-MB-468 cell lines were treated with or without MG132 (10 μM) for 12 h. The expression of DHODH and USP24 proteins was assessed by Western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. ns no significance.

Article Snippet: USP5 (10473-1-AP), USP9X (81892-1-RR), USP10 (19374-1-AP), USP13 (16840-1-AP), USP24 (13126-1-AP), USP28 (17707-1-AP), USP38 (17767-1-AP), USP48 (12076-1-AP), USP53 (83846-1-RR), DHODH (14877-1-AP), GSS (15712-1-AP), ACSL4 (22401-1-AP), NRF2 (80593-1-AP), Ki67 (27309-1-AP), and FSP1 (20886-1-AP) antibodies were purchased from Proteintech (Chicago, USA).

Techniques: Western Blot, Stable Transfection, Transfection, Control, shRNA, Expressing

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP24 suppresses ferroptosis in triple-negative breast cancer by stabilizing DHODH protein

doi: 10.1038/s41419-025-07895-4

Figure Lengend Snippet: A , B Endogenous USP24 and DHODH interactions were detected by coimmunoprecipitation using USP24 and DHODH antibodies, respectively, in MDA-MB-231 and MDA-MB-468 cells. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. C , D The intracellular localization of USP24 (red) and DHODH (green) in MDA-MB-231 and MDA-MB-468 cells was examined by immunofluorescence staining using USP24 and DHODH antibodies and visualized by fluorescence microscopy. DAPI was used to stain nuclei. Scale bars: 5 μm. Immunofluorescence colocalization was quantified using the Manders’ coefficient. E , F The indicated MDA-MB-231 cell lines were pretreated with MG132 (10 μM) for 24 h, and then the extracts were immunoprecipitated with anti-DHODH antibodies and immunoblotted with anti-ubiquitin (Ub), anti-K48-linked ubiquitin, and anti-DHODH antibodies. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. * P < 0.05, **** P < 0.0001. ns no significance. G , H MDA-MB-231 and MDA-MB-468 cells stably transfected with USP24 shRNA#1 or control shRNA were stably expressed with Flag-DHODH or control vector. USP24 and DHODH expression in cells was measured by Western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. * P < 0.05, *** P < 0.001, **** P < 0.0001. ns no significance. I Cell death was assessed using propidium iodide (PI) staining in the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with the indicated doses of RSL3 for 24 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns no significance. J , K Migration ( J ) and cloning formation ( K ) abilities of the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with RSL3 (0.5 μM for MDA-MB-231 cells; 0.25 μM for MDA-MB-468 cells) for 6 h. Mean ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns no significance. L Lipid peroxidation of the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with RSL3 (0.5 μM for MDA-MB-231 cells; 0.25 μM for MDA-MB-468 cells) for 6 h. Mean ± SD, n = 3. ** P < 0.01, **** P < 0.0001. ns no significance. M Cell death was assessed using propidium iodide (PI) staining in the indicated MDA-MB-231 and MDA-MB-468 cell lines treated with RSL3 (0.5 μM for MDA-MB-231 cells; 0.25 μM for MDA-MB-468 cells) in the presence or absence of MitoQH 2 (2.5 μM) for 24 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: USP5 (10473-1-AP), USP9X (81892-1-RR), USP10 (19374-1-AP), USP13 (16840-1-AP), USP24 (13126-1-AP), USP28 (17707-1-AP), USP38 (17767-1-AP), USP48 (12076-1-AP), USP53 (83846-1-RR), DHODH (14877-1-AP), GSS (15712-1-AP), ACSL4 (22401-1-AP), NRF2 (80593-1-AP), Ki67 (27309-1-AP), and FSP1 (20886-1-AP) antibodies were purchased from Proteintech (Chicago, USA).

Techniques: Immunofluorescence, Staining, Fluorescence, Microscopy, Immunoprecipitation, Ubiquitin Proteomics, Stable Transfection, Transfection, shRNA, Control, Plasmid Preparation, Expressing, Western Blot, Migration, Cloning

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP24 suppresses ferroptosis in triple-negative breast cancer by stabilizing DHODH protein

doi: 10.1038/s41419-025-07895-4

Figure Lengend Snippet: A Schematic of the anticancer effect of RSL3 (2 mg/kg/2 days) in the indicated MDA-MB-231 tumor xenograft models. B The tumor volume of each group was calculated every 2 days. C The excised tumors were photographed on day 22. D The tumor weights of each group. Mean ± SD, n = 6. * P < 0.05, *** P < 0.001, **** P < 0.0001. E The mice body weight of each group was recorded every 2 days. F The expression of USP24, DHODH, Ki67, 4-HNE, and cleaved-caspase 3 were detected by immunohistochemical staining. Representative images and quantification in each group were shown (n = 3 samples). Scale bar, 20 μm. Mean ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns no significance.

Article Snippet: USP5 (10473-1-AP), USP9X (81892-1-RR), USP10 (19374-1-AP), USP13 (16840-1-AP), USP24 (13126-1-AP), USP28 (17707-1-AP), USP38 (17767-1-AP), USP48 (12076-1-AP), USP53 (83846-1-RR), DHODH (14877-1-AP), GSS (15712-1-AP), ACSL4 (22401-1-AP), NRF2 (80593-1-AP), Ki67 (27309-1-AP), and FSP1 (20886-1-AP) antibodies were purchased from Proteintech (Chicago, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP24 suppresses ferroptosis in triple-negative breast cancer by stabilizing DHODH protein

doi: 10.1038/s41419-025-07895-4

Figure Lengend Snippet: A Schematic of the anticancer effect of RSL3 (2 mg/kg/2 days) with or without WP1130 (25 mg/kg/2 days) in the MDA-MB-231 tumor xenograft models. B The tumor volume of each group was calculated every 2 days. C The excised tumors were photographed on day 22. D The tumor weights of each group. Mean ± SD, n = 6. **** P < 0.0001. E The mice body weight of each group was recorded every 2 days. F The expression of USP24, DHODH, Ki67, 4-HNE, and cleaved-caspase 3 were detected by immunohistochemical staining. Representative images and quantification in each group were shown (n = 3 samples). Scale bar, 20 μm. Mean ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns no significance.

Article Snippet: USP5 (10473-1-AP), USP9X (81892-1-RR), USP10 (19374-1-AP), USP13 (16840-1-AP), USP24 (13126-1-AP), USP28 (17707-1-AP), USP38 (17767-1-AP), USP48 (12076-1-AP), USP53 (83846-1-RR), DHODH (14877-1-AP), GSS (15712-1-AP), ACSL4 (22401-1-AP), NRF2 (80593-1-AP), Ki67 (27309-1-AP), and FSP1 (20886-1-AP) antibodies were purchased from Proteintech (Chicago, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP24 suppresses ferroptosis in triple-negative breast cancer by stabilizing DHODH protein

doi: 10.1038/s41419-025-07895-4

Figure Lengend Snippet: USP24 protects TNBC from ferroptosis by stabilizing DHODH in TNBC cells. USP24 blockade is synthetic lethal with GPX4 inhibitors, inducing ferroptosis through a DHODH-dependent mechanism.

Article Snippet: USP5 (10473-1-AP), USP9X (81892-1-RR), USP10 (19374-1-AP), USP13 (16840-1-AP), USP24 (13126-1-AP), USP28 (17707-1-AP), USP38 (17767-1-AP), USP48 (12076-1-AP), USP53 (83846-1-RR), DHODH (14877-1-AP), GSS (15712-1-AP), ACSL4 (22401-1-AP), NRF2 (80593-1-AP), Ki67 (27309-1-AP), and FSP1 (20886-1-AP) antibodies were purchased from Proteintech (Chicago, USA).

Techniques: