Journal: Cells

Article Title: DCUN1D1 Is an Essential Regulator of Prostate Cancer Proliferation and Tumour Growth That Acts through Neddylation of Cullin 1, 3, 4A and 5 and Deregulation of Wnt/Catenin Pathway.

doi: 10.3390/cells12151973

Figure Lengend Snippet: Figure 5. Blockage of DCUN1D1 decreases global neddylation, ubiquitination and expression of neddylation components and shows preferential neddylation activity of cullin proteins in PCa. Western blot analysis of DU145 and DU145 DCUN1D1-KD cells. Protein extracts were obtained from the cells and subjected to Western blot analysis using (a) anti-ubiquitin, (b) anti-Nedd8. Blockage of DCUN1D1 decreased the expression of the neddylation pathway components including (c) the E1 NAE heterodimer APPB1 (top panel) and the neddylation-conjugating enzyme, UBC12 (bottom panel) and (d) the cullin-associated proteins RBX1 (top panel) and CAND1 (bottom panel). Inhibition of DCUN1D1 showed preferential NEDD8 modification of the cullin family of proteins. Immunoblot analysis of DU145 and DU145 DCUN1D1 knockdown cell protein extracts using (e) anti-cullin 1, (f) anti-cullin 3, (g) anti-cullin 4A, (h) anti-cullin 4B and (i) anti-cullin 5. The GAPDH loading control was probed using the anti-GAPDH antibody. Experiments were independently repeated three times and a representative image of an independent experiment is represented.

Article Snippet: The following primary antibodies were used: DCUN1D1 (Sigma HPA035911), Nedd8 (Cell Signaling Technology, Danver, MA, USA CST2745S), Ubiquitin (Cell Signaling Technology CST39365), cullin 1 (Cell Signaling Technology CST4995), cullin 2 (Novus Biologicals, Centennial, CO, USA NBP1-67535), cullin 3 (Cell Signaling Technology CST2759S), cullin 4A (Cell Signaling Technology CST2699S), cullin 4B (Bio-Rad VMA00360) Cells 2023, 12, 1973 6 of 22 cullin 5 (Novus Biologicals NBP1-22970), APPBP1 (Cell Signaling Technology CST14321), UBA3 (Novus Biologicals NBP2-48628), UBC12 (Novus Biologicals NBP1-31459), CAND1 (Cell Signaling Technology CST8759S), RBX1 (Novus Biologicals NBP2-20113), β-Catenin (Cell Signaling Technology CST4394S) phosphor-β-Catenin (Cell Signaling Technology CST2009S) and Lef1 (Santa Cruz sc-374522).

Techniques: Ubiquitin Proteomics, Expressing, Activity Assay, Western Blot, Inhibition, Knockdown, Control

Journal: Biochemical and biophysical research communications

Article Title: Disruption of protein neddylation with MLN4924 attenuates paclitaxel-induced apoptosis and microtubule polymerization in ovarian cancer cells.

doi: 10.1016/j.bbrc.2018.12.048

Figure Lengend Snippet: Fig. 4. UBE2M and UBE2F knockdown attenuated the effects of PTX in HO8910 cells. (A) HO8910 cells infected with retroviruses expressing shRNAs for control, UBE2M, or UBE2F were selected in puromycin and analyzed for UBE2M and UBE2F expression by western blotting. (B) The numbers of surviving cells were counted for different periods of time after DMSO or PTX (0.05 mM) treatment. (C) HO8910 cells with stable knockdown of UBE2M or UBE2F were stained for a-tubulin at 48 h after treatment with 0.05 mM PTX. Overlay of blue (nuclei) and red (a-tubulin) channels is shown. Scale bars: 20 mm. (D) Levels of a-tubulin protein were further confirmed by western blotting with GAPDH and b-actin as loading control.

Article Snippet: To generate UBE2M or UBE2F stable knockdown cell lines, we used UBE2M- or UBE2F-specific shRNA plasmids co-transfected with pRSV-Rev (cat. no. 12253; Addgene), pMDLg/pRRE (cat. no. 12251; Addgene), and pCMV-VSV-G (cat. no. 8454; Addgene) into HEK293Tcells.

Techniques: Knockdown, Infection, Expressing, Control, Western Blot, Staining

Journal: Translational Cancer Research

Article Title: Overexpression of UBE2M through Wnt/β-Catenin signaling is associated with poor prognosis and chemotherapy resistance in colorectal cancer

doi: 10.21037/tcr-20-2641

Figure Lengend Snippet: UBE2M expression in different tissues. (A) Expression of UBE2M in normal tissues and CRC tissues according to the relative mRNA levels of UBE2M gene measured by quantitative real-time PCR. (B) Expression of UBE2M in CRC tissues at different tumor stages. (C,D) Expression of UBE2M in colon and rectum cancer obtained from TCGA database. Statistical significance was determined by t test. ***, P<0.001. UBE2M, ubiquitin-conjugating enzyme E2 M; CRC, colorectal carcinoma; mRNA, messenger RNA; PCR, polymerase chain reaction; TCGA, The Cancer Genome Atlas.

Article Snippet: Full-length human UBE2M was cloned into peroxisomal biogenesis factor 3 (pEX-3) vector to construct plasmid for the overexpression of UBE2M (Biosyntech, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics, Polymerase Chain Reaction

Journal: Translational Cancer Research

Article Title: Overexpression of UBE2M through Wnt/β-Catenin signaling is associated with poor prognosis and chemotherapy resistance in colorectal cancer

doi: 10.21037/tcr-20-2641

Figure Lengend Snippet: Expression and location of UBE2M protein in normal tissues and CRC tissues according to 2 pairs of representative clinical specimens. Scale bars =50 µm. UBE2M, ubiquitin-conjugating enzyme E2 M; CRC, colorectal carcinoma.

Article Snippet: Full-length human UBE2M was cloned into peroxisomal biogenesis factor 3 (pEX-3) vector to construct plasmid for the overexpression of UBE2M (Biosyntech, China).

Techniques: Expressing, Ubiquitin Proteomics

Journal: Translational Cancer Research

Article Title: Overexpression of UBE2M through Wnt/β-Catenin signaling is associated with poor prognosis and chemotherapy resistance in colorectal cancer

doi: 10.21037/tcr-20-2641

Figure Lengend Snippet: Kaplan-Meier curves comparing the overall survival and disease-free survival of CRC patients with low and high expression of UBE2M. CRC, colorectal carcinoma; UBE2M, ubiquitin-conjugating enzyme E2 M.

Article Snippet: Full-length human UBE2M was cloned into peroxisomal biogenesis factor 3 (pEX-3) vector to construct plasmid for the overexpression of UBE2M (Biosyntech, China).

Techniques: Expressing, Ubiquitin Proteomics

Journal: Translational Cancer Research

Article Title: Overexpression of UBE2M through Wnt/β-Catenin signaling is associated with poor prognosis and chemotherapy resistance in colorectal cancer

doi: 10.21037/tcr-20-2641

Figure Lengend Snippet: Multivariable cox regression analysis of risk factors of overall survival in CRC patients

Article Snippet: Full-length human UBE2M was cloned into peroxisomal biogenesis factor 3 (pEX-3) vector to construct plasmid for the overexpression of UBE2M (Biosyntech, China).

Techniques: Over Expression

Journal: Translational Cancer Research

Article Title: Overexpression of UBE2M through Wnt/β-Catenin signaling is associated with poor prognosis and chemotherapy resistance in colorectal cancer

doi: 10.21037/tcr-20-2641

Figure Lengend Snippet: HCT116, HCT116/5-FU, HCT116/oxaliplatin, SW480, SW480/5-FU, and SW480/oxaliplatin cells were treated with an increased dose of 5-FU and oxaliplatin for 72 h. Cell viability was determined according to CellTiter-Glo Luminescent Cell Viability Assay. (E) Protein levels of UBE2M in HCT116, HCT116/5-FU, HCT116/oxaliplatin, SW480, SW480/5-FU, and SW480/oxaliplatin cells were measured by western blot. (F) Quantitative representation of the band intensities. Statistical significance was determined by t test. ***, P<0.01. UBE2M, ubiquitin-conjugating enzyme E2 M.

Article Snippet: Full-length human UBE2M was cloned into peroxisomal biogenesis factor 3 (pEX-3) vector to construct plasmid for the overexpression of UBE2M (Biosyntech, China).

Techniques: Cell Viability Assay, Western Blot, Ubiquitin Proteomics

Journal: Translational Cancer Research

Article Title: Overexpression of UBE2M through Wnt/β-Catenin signaling is associated with poor prognosis and chemotherapy resistance in colorectal cancer

doi: 10.21037/tcr-20-2641

Figure Lengend Snippet: The expression of UBE2M regulated chemotherapy resistance via the activation of β-catenin. (A) Protein levels of UBE2M in HCT116, HCT116 + siRNA, HCT116 + plasmid, SW480, SW480 + siRNA, and SW480 + plasmid cells were measured by western blot. (B) Quantitative representation of the band intensities. (C,D,E,F) HCT116, HCT116 + siRNA, HCT116 + plasmid, HCT116/5-FU, HCT116/5-FU + siRNA, HCT116/oxaliplatin, and HCT116/oxaliplatin + siRNA cells were treated with an increased dose of 5-FU and oxaliplatin for 72 h. Cell viability was determined according to CellTiter-Glo Luminescent Cell Viability Assay. (G) Protein levels of β-catenin in HCT116, HCT116 + siRNA, HCT116 + plasmid, SW480, SW480 + siRNA, and SW480 + plasmid cells were measured by western blot. (H) Quantitative representation of the band intensities. Statistical significance was determined by t test. ***, P<0.01. UBE2M, ubiquitin-conjugating enzyme E2 M; siRNA, small interfering ribonucleic acid.

Article Snippet: Full-length human UBE2M was cloned into peroxisomal biogenesis factor 3 (pEX-3) vector to construct plasmid for the overexpression of UBE2M (Biosyntech, China).

Techniques: Expressing, Activation Assay, Plasmid Preparation, Western Blot, Cell Viability Assay, Ubiquitin Proteomics