Journal: International journal of cancer

Article Title: Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

doi: 10.1002/ijc.34769

Figure Lengend Snippet: FIGURE 1 Limited primary tumor growth and less lung metastasis in mice carrying inducible Ubr5-knockdown 4T1 cells. (A) Schematic diagram of the experimental design. 5 105 4T1-GFP, 4T1-Tet-shUbr5 cells are injected into the mammary gland of female BALB/c mice and on D3, the mice are treated with 100 mg/kg Dox by gavage daily. On day 27, mice are sacrificed, and tumors are dissected. (B) 4T1 tumor growth curve. (C) Tumor weight is measured. (D) Ubr5 mRNA expression of mice bearing tumor is detected by real-time quantitative PCR. (E). Protein level of UBR5 in tumor analyzed by Western blot. (F) Representative UBR5 IHC staining of tumor from mice bearing 4T1/GFP, 4T1/Tet-shUbr5 cells. (G) Statistical analysis of UBR5 immunostaining IRS score. (H) Palpable metastatic nodules on lung surfaces are enumerated. Data represents mean ± SEM; **P < .01; ***P < .001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To generate 4T1/Ubr5 / + hUBR5 and 4T1/Ubr5 / + C2768A cell lines, pCMV-Tag2B UBR5 and pCMV-Tag2B C2768A (Addgene, #37188 and #37189, respectively) were transfected into 4T1/Ubr5 / cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA).

Techniques: Knockdown, Injection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Immunostaining

Journal: International journal of cancer

Article Title: Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

doi: 10.1002/ijc.34769

Figure Lengend Snippet: FIGURE 2 Inducible Ubr5 expression knockdown suppresses post-surgery lung metastasis. (A) Schematic for removing primary tumor and knocking down Ubr5 expression with daily Dox addition (100 mg/kg) by gavage post-surgery in mice. (B, C) Representative images for lung nodules stained with India ink (B) Lung nodules in tumor-bearing are quantified (C). (D, E) The 6-thioguanine clonogenicity assay of Tet-scr and Tet-shUbr5 tumors (on D35) for lung metastasis is conducted. Images of representative experiment are depicted (D) and colonies are quantified (E). (F) Overall survival rates of the mice (Tet-scr, n = 8; Tet-scr(+Dox), n = 6; Tet-shUbr5, n = 5; Tet-shUbr5(+Dox), n = 5). (G) UBR5 protein expression in hUBR5- and C2768A-reconstituted 4T1/Ubr5/ cells is evaluated in Western blotting. (H) Representative result of E2 discharge assay for UBR5 and C2768A. (I) Quantitation of the discharged E2-Ub by UBR5 and C2768A. (J) A total of 5 105 Tet-scr, Tet-shUbr5, Ubr5/ + hUBR5 or Ubr5/ + C2768A cells are injected into 6- to 8-week-old female BALB/c mice (with 8, 8, 8 and 16 mice, respectively, for the four cell lines). Tumor volume is measured on D20. (K) The metastatic cells in the lung are enumerated by the 6-thiogaunine assay. (L) After removing the primary tumor, the mice with similar primary tumor volumes are selected from all groups and administrated with Dox daily by gavage (100 mg/kg). On D30, the mice are sacrificed. Lung metastatic nodules are stained with Indian ink and quantified (Tet-scr, n = 5; Tet-shUbr5, n = 5; Ubr5/ + hUBR5, n = 5, Ubr5/ + C2768A, n = 4). Data represents mean ± SEM; *P < .05; **P < .01; ***P < .001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To generate 4T1/Ubr5 / + hUBR5 and 4T1/Ubr5 / + C2768A cell lines, pCMV-Tag2B UBR5 and pCMV-Tag2B C2768A (Addgene, #37188 and #37189, respectively) were transfected into 4T1/Ubr5 / cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA).

Techniques: Expressing, Knockdown, Staining, Western Blot, Quantitation Assay, Injection

Journal: International journal of cancer

Article Title: Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

doi: 10.1002/ijc.34769

Figure Lengend Snippet: FIGURE 4 UBR5 suppresses apoptosis and promotes lung metastasis via targeting CDC73. (A) UBR5 and CDC73 protein expression in 4T1 cells with Dox-induced shUbr5 and shCdc73 expression. (B) Representative FACS images of Dox-treated Tet-scr, Tet-shUbr5, Tet-shCdc73 and Tet-shUbr5-shCdc73 cells exposed to Dovitinib (1 μm) for 24 h. (C) Quantitation of apoptotic cells in b. (D) Apoptosis in lung sections of mice bearing Dox-treated Tet-scr, Tet-shUbr5, Tet-shCdc73 or Tet-shUbr5-shCdc73 tumors, respectively, is evaluated by TUNEL staining on D35 (n = 3 mice per group), Scale bar: 100 μm. (E) Representative IHC staining for Ki67 expression images in the lung with quantifications. (F) Lung metastatic nodules in tumor-bearing are stained with India ink and quantified (5 mice/group). (G) Overall survival rates of mice carrying Dox- treated Tet-scr (n = 7), Tet-shUbr5 (n = 5), Tet-shCdc73 (n = 6) or Tet-shUbr5-shCdc73 (n = 8) tumors. Data represents mean ± SEM; *P < .05; **P < .01; ***P < .001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To generate 4T1/Ubr5 / + hUBR5 and 4T1/Ubr5 / + C2768A cell lines, pCMV-Tag2B UBR5 and pCMV-Tag2B C2768A (Addgene, #37188 and #37189, respectively) were transfected into 4T1/Ubr5 / cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA).

Techniques: Expressing, Quantitation Assay, TUNEL Assay, Staining, Immunohistochemistry

Journal: International journal of cancer

Article Title: Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

doi: 10.1002/ijc.34769

Figure Lengend Snippet: FIGURE 6 Effects of human TNBC-expressed UBR5 on tumor growth and lung metastases. (A) IHC staining of UBR5 and CDC73 proteins in primary tumors from TNBC patients (n = 70 samples). Scale bars: 200 μm. (B) Correlation between tumor UBR5 and CDC73 expression levels in human TNBC patients. (C) mRNA levels of CDC73 in primary breast cancer tissues (n = 168) and metastatic sites (n = 52). (D) Metastatic nodules in the lung and liver of tumor-bearing mice are quantified on D45. (E) Representative IHC staining of UBR5 and CDC73 in lung metastatic nodules. Scale bar: 100 μm. (F) UBR5 and CDC73 protein expression in MDA-MB-231-Vector, MDA-MB-231-shUBR5 or MDA-MB- 231-shUBR5-shCDC73 cell lines. (G) Tumor growth rates of NSG mice. (H) Metastatic nodules in the lung and liver of tumor-bearing mice are quantified on D38. Data represents mean ± SEM; *P < .05; **P < .01; ***P < .001. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: To generate 4T1/Ubr5 / + hUBR5 and 4T1/Ubr5 / + C2768A cell lines, pCMV-Tag2B UBR5 and pCMV-Tag2B C2768A (Addgene, #37188 and #37189, respectively) were transfected into 4T1/Ubr5 / cells using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA).

Techniques: Immunohistochemistry, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a Immunoblots of UBR5 protein in wild type (WT) and three UBR5 −/− (made with unique guide RNA) in 2fTGH-ISRE-Luc cells. Quantification of the ( b ) intracellular ISRE-Luc activity, c secreted IFN-β protein by ELISA, d cellular IFNB1 mRNA levels in 2fTGH-ISRE-Luc cells transfected with poly (I:C) for 12 h. Quantification of the ( e ) intracellular ISRE-Luc activity, f secreted IFN-β protein, g cellular IFNB1 mRNA, h secreted type I IFNs (Bioassay) in WT, UBR5 −/− (made with gRNA #1) and IFIH1 −/− (gene symbol for MDA5) HEK293T cells transfected with poly (I:C) for 12 h. i Quantification of ISRE-Luc activity in 2fTGH-ISRE-Luc cells transfected with a negative control siRNA (siCtrl) or UBR5 siRNA for 48 h, then poly (I:C) for 12 h. j Measurement of the Luc activity in HEK293T cells transfected with a pcDNA3.1 vector or UBR5 expression plasmid, together with an IFNB1 promoter-driven firefly luciferase (Luc) and an ACTIN promoter-driven renilla Luc plasmid (internal control) for 24 h. The cells were then transfected with poly (I:C) for 12 h. k Quantification of the IFNB1 mRNA in HEK293T cells transfected with a vector or UBR5 expression plasmid for 24 h, then poly (I:C) for 12 h. l Immunoblots of UBR5 protein in mouse primary embryonic fibroblasts (MEF) or bone marrow-derived macrophages. Quantification of ( m ) the Ifnb1 mRNA in MEFs, secreted IFN-β protein in ( n ) MEFs and ( o ) macrophages transfected with poly (I:C). Quantification of the ( p ) Ifnb1 mRNA levels, q secreted IFN-β protein in MEFs transfected with 5-ppp hpRNA (RIG-I agonist). Ubr5 iKO : Ubr5 inducible knockout by Tamoxifen. Data presented in b – d , g – i : mean ± S.E.M, ordinary one-way ANOVA with Dunnett’s test, n = 3 biological independent experiments; for b : **** p < 0.0001, *** p = 0.0003, * p = 0.0305 vs WT; for c : **** p < 0.0001 vs WT; for d : *** p = 0.0003, ** p = 0.0022 vs WT; for g : *** p = 0.0001, **** p < 0.0001 vs WT; for h : **** p < 0.0001 vs WT; for i : **** p < 0.0001, *** p = 0.0005 vs siCtrl. Mult i plicity adjusted p values are p resented. Data presented in e , f , j , k , m – q : mean ± S.E.M, two-tailed student’s t test; for e : *** p = 0.0007, **** p < 0.0001, n = 4 biological independent experiments; for f : *** p = 0.0002, * p = 0.012; for j : ** p = 0.0015, ** p = 0.0018 in sequence; for k : ** p = 0.0014, ** p = 0.0084 in sequence; for m : * p = 0.0224, **** p < 0.0001; for n : ** p = 0.0024, * p = 0.0407; for o : * p = 0.0478, * p = 0.0129, *** p = 0.0002 in sequence; for p : *** p = 0.0005, *** p = 0.0001 in sequence; for q : * p = 0.0263, * p = 0.0138 in sequence; n = 3 biological independent experiments in f , j , k , m – q . Adjusted p values are presented. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Bioassay, Negative Control, Plasmid Preparation, Expressing, Luciferase, Control, Derivative Assay, Knock-Out, Two Tailed Test, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a Immunoblots of UBR5 protein in wild type (WT) and three UBR5 −/− (made with unique guide RNA) in 2fTGH-ISRE-Luc cells. Quantification of the ( b ) intracellular ISRE-Luc activity, c secreted IFN-β protein by ELISA, d cellular IFNB1 mRNA levels in 2fTGH-ISRE-Luc cells transfected with poly (I:C) for 12 h. Quantification of the ( e ) intracellular ISRE-Luc activity, f secreted IFN-β protein, g cellular IFNB1 mRNA, h secreted type I IFNs (Bioassay) in WT, UBR5 −/− (made with gRNA #1) and IFIH1 −/− (gene symbol for MDA5) HEK293T cells transfected with poly (I:C) for 12 h. i Quantification of ISRE-Luc activity in 2fTGH-ISRE-Luc cells transfected with a negative control siRNA (siCtrl) or UBR5 siRNA for 48 h, then poly (I:C) for 12 h. j Measurement of the Luc activity in HEK293T cells transfected with a pcDNA3.1 vector or UBR5 expression plasmid, together with an IFNB1 promoter-driven firefly luciferase (Luc) and an ACTIN promoter-driven renilla Luc plasmid (internal control) for 24 h. The cells were then transfected with poly (I:C) for 12 h. k Quantification of the IFNB1 mRNA in HEK293T cells transfected with a vector or UBR5 expression plasmid for 24 h, then poly (I:C) for 12 h. l Immunoblots of UBR5 protein in mouse primary embryonic fibroblasts (MEF) or bone marrow-derived macrophages. Quantification of ( m ) the Ifnb1 mRNA in MEFs, secreted IFN-β protein in ( n ) MEFs and ( o ) macrophages transfected with poly (I:C). Quantification of the ( p ) Ifnb1 mRNA levels, q secreted IFN-β protein in MEFs transfected with 5-ppp hpRNA (RIG-I agonist). Ubr5 iKO : Ubr5 inducible knockout by Tamoxifen. Data presented in b – d , g – i : mean ± S.E.M, ordinary one-way ANOVA with Dunnett’s test, n = 3 biological independent experiments; for b : **** p < 0.0001, *** p = 0.0003, * p = 0.0305 vs WT; for c : **** p < 0.0001 vs WT; for d : *** p = 0.0003, ** p = 0.0022 vs WT; for g : *** p = 0.0001, **** p < 0.0001 vs WT; for h : **** p < 0.0001 vs WT; for i : **** p < 0.0001, *** p = 0.0005 vs siCtrl. Mult i plicity adjusted p values are p resented. Data presented in e , f , j , k , m – q : mean ± S.E.M, two-tailed student’s t test; for e : *** p = 0.0007, **** p < 0.0001, n = 4 biological independent experiments; for f : *** p = 0.0002, * p = 0.012; for j : ** p = 0.0015, ** p = 0.0018 in sequence; for k : ** p = 0.0014, ** p = 0.0084 in sequence; for m : * p = 0.0224, **** p < 0.0001; for n : ** p = 0.0024, * p = 0.0407; for o : * p = 0.0478, * p = 0.0129, *** p = 0.0002 in sequence; for p : *** p = 0.0005, *** p = 0.0001 in sequence; for q : * p = 0.0263, * p = 0.0138 in sequence; n = 3 biological independent experiments in f , j , k , m – q . Adjusted p values are presented. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Bioassay, Negative Control, Plasmid Preparation, Expressing, Luciferase, Control, Derivative Assay, Knock-Out, Two Tailed Test, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a Immunoblots of UBR5 protein in wild type (WT) and three UBR5 −/− (made with unique guide RNA) in 2fTGH-ISRE-Luc cells. Quantification of the ( b ) intracellular ISRE-Luc activity, c secreted IFN-β protein by ELISA, d cellular IFNB1 mRNA levels in 2fTGH-ISRE-Luc cells transfected with poly (I:C) for 12 h. Quantification of the ( e ) intracellular ISRE-Luc activity, f secreted IFN-β protein, g cellular IFNB1 mRNA, h secreted type I IFNs (Bioassay) in WT, UBR5 −/− (made with gRNA #1) and IFIH1 −/− (gene symbol for MDA5) HEK293T cells transfected with poly (I:C) for 12 h. i Quantification of ISRE-Luc activity in 2fTGH-ISRE-Luc cells transfected with a negative control siRNA (siCtrl) or UBR5 siRNA for 48 h, then poly (I:C) for 12 h. j Measurement of the Luc activity in HEK293T cells transfected with a pcDNA3.1 vector or UBR5 expression plasmid, together with an IFNB1 promoter-driven firefly luciferase (Luc) and an ACTIN promoter-driven renilla Luc plasmid (internal control) for 24 h. The cells were then transfected with poly (I:C) for 12 h. k Quantification of the IFNB1 mRNA in HEK293T cells transfected with a vector or UBR5 expression plasmid for 24 h, then poly (I:C) for 12 h. l Immunoblots of UBR5 protein in mouse primary embryonic fibroblasts (MEF) or bone marrow-derived macrophages. Quantification of ( m ) the Ifnb1 mRNA in MEFs, secreted IFN-β protein in ( n ) MEFs and ( o ) macrophages transfected with poly (I:C). Quantification of the ( p ) Ifnb1 mRNA levels, q secreted IFN-β protein in MEFs transfected with 5-ppp hpRNA (RIG-I agonist). Ubr5 iKO : Ubr5 inducible knockout by Tamoxifen. Data presented in b – d , g – i : mean ± S.E.M, ordinary one-way ANOVA with Dunnett’s test, n = 3 biological independent experiments; for b : **** p < 0.0001, *** p = 0.0003, * p = 0.0305 vs WT; for c : **** p < 0.0001 vs WT; for d : *** p = 0.0003, ** p = 0.0022 vs WT; for g : *** p = 0.0001, **** p < 0.0001 vs WT; for h : **** p < 0.0001 vs WT; for i : **** p < 0.0001, *** p = 0.0005 vs siCtrl. Mult i plicity adjusted p values are p resented. Data presented in e , f , j , k , m – q : mean ± S.E.M, two-tailed student’s t test; for e : *** p = 0.0007, **** p < 0.0001, n = 4 biological independent experiments; for f : *** p = 0.0002, * p = 0.012; for j : ** p = 0.0015, ** p = 0.0018 in sequence; for k : ** p = 0.0014, ** p = 0.0084 in sequence; for m : * p = 0.0224, **** p < 0.0001; for n : ** p = 0.0024, * p = 0.0407; for o : * p = 0.0478, * p = 0.0129, *** p = 0.0002 in sequence; for p : *** p = 0.0005, *** p = 0.0001 in sequence; for q : * p = 0.0263, * p = 0.0138 in sequence; n = 3 biological independent experiments in f , j , k , m – q . Adjusted p values are presented. Source data are provided as a Source Data file.

Article Snippet: FLAG-TRIM28 (Item #124960) , pcDNA3 Myc-Sumo3 WT (Item #48964), pcDNA3 HA-Sumo1 WT (Item #48966), pcDNA3 HA-Sumo2 WT (Item #48967 ) , GFP-UBR5 ∆HECT (Item #52051) , pET28-His6-Ubc9 (Item #133909) , pCMV-Tag2B UBR5 (Item #37188), pCMV-Tag2B UBR5 C2768A (Item #37189), and pEGFP-C1-UBR5 (Item #37190) were obtained from Addgene Inc. (Watertown, MA, United States).

Techniques: Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Bioassay, Negative Control, Plasmid Preparation, Expressing, Luciferase, Control, Derivative Assay, Knock-Out, Two Tailed Test, Sequencing

Journal: Oncogene

Article Title: Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin

doi: 10.1038/onc.2016.336

Figure Lengend Snippet: UBR5-containing EDVP E3 ligase complex interacts and regulates MOAP-1 ubiquitylation and stability. ( a ) Flag-MOAP-1 was transfected into 293T cells, and lysates were prepared 48 h posttransfection. Co-IP with Flag M2 agarose beads were performed and immunoblotted with antibodies as indicated. n =3 independent experiments. ( b ) In vitro ubiquitylation assay was performed as in the presence or absence of recombinant DDB1, VprBP and Dyrk2. n =3 independent experiments. Original uncropped image is shown in . ( c ) siCtrl or siDyrk2 was transfected into H1299 or 293T cells, and Flag-MOAP-1 was transfected into each siRNA transfectant 24 h post-siRNA transfection. Cell lysates were prepared under denaturing condition; Flag-MOAP-1 was immunoprecipitated and immunoblotted with ubiquitin antibody. Same membrane was re-blotted with Flag antibody for Flag-MOAP-1. n =3 independent experiments. Asterisk in Dyrk2 blot indicates non-specific band. Quantification of MOAP-1 ubiquitylation is shown in . ( d ) 293T cells transfected with siCtrl, siDyrk2 or siUBR5 were treated with CHX 48 h posttransfection. Cells were collected at the indicated times after CHX treatment, and lysates were prepared and immunoblotted as indicated (left). MOAP-1 protein level was quantified and plotted (right). The MOAP-1 abundance at 0 time point was set at 100%. n =4 independent experiments (means±s.e.m.). Molecular weight markers are in kDa.

Article Snippet: MOAP-1 expression constructs were described previously. pCMV-Tag2B EDD, which encodes Flag-UBR5, was a gift from Darren Saunders and Charles Watts (Addgene, Cambridge, MA, USA, plasmid no. 37188). pDEST12.2 DYRK2 WT, which encodes Flag-Dyrk2, was a gift from Anjana Rao (Addgene plasmid no. 20005).

Techniques: Transfection, Co-Immunoprecipitation Assay, In Vitro, Ubiquitin Assay, Recombinant, Immunoprecipitation, Ubiquitin Proteomics, Membrane, Molecular Weight