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: bioRxiv

Article Title: N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

doi: 10.1101/2022.09.01.505523

Figure Lengend Snippet: (A) MS-spectra for the N-terminal peptide of endogenous IST1 (P53990) from HAP1 WT and NatC KO cells following trypsin digestion and strong cation exchange (SCX) enrichment. (B) Bar graph showing the degree of Nt-acetylation of IST1 in HAP1 WT and NatC KO cells as determined by proteomics. Data are shown as mean ± SD (n = 4). (C) Endogenous IST1 protein levels from indicated HAP1 cells determined by immunoblotting. (D) Immunoblot analysis of confirmed and putative NatC substrates using total cell extract from HAP1 WT and NatC KO cells. (E) N-terminal variants of UBE2M-FLAG were expressed in HAP1 NAA30 -KO cells and protein levels were determined by immunoblotting. (F) HAP1 WT and NAA30 -KO cells were transfected with the indicated UBE2M-V5-P2A-GST-GFP reporter construct, and protein levels were determined by immunoblot analysis. UBE2M-V5 levels were normalized to GST-GFP and expressed relative to WT sample. Data are shown as mean ± SD of four independent experiments. ***p < 0.0004; two-tailed unpaired t test. (G) NAA30-WT-V5 and NAA30-mut-V5 was immunoprecipitated from HeLa cell extracts and used in Nt-acetylation assays with [ 14 C]-acetyl-CoA and synthetic peptides representing the NatC substrates UBE2M (MIKL) and ARFRP1 (MYTL), and the NAA80/NatH substrate β-actin (DDDI). The experiment was performed three independent times with three technical replicates each. Data of one representative setup is shown as mean ± SD. (H) NatC regulates the protein level of UBE2M, UBE2F ARFRP1, and CAPNS1. Immunoblot analysis of HAP1 WT and NAA30 -KO cells transfected with control V5 plasmid, NAA30-V5 or the catalytically dead mutant NAA30-mut-V5. (I) HAP1 WT and NAA30 -KO cells were treated with proteasomal (MG132 and bortezomib (BMZ)) and lysosomal inhibitors (bafilomycin A (BafA), leupeptin (LP) or ammonium chloride (NH4Cl)) for 6 h followed by immunoblot analysis using the indicated antibodies. DMSO served as vehicle control.

Article Snippet: The plasmid pCMV6-UBE2M-Myc-DDK (DDK is the same as FLAG®) was obtained from OriGene Technologies (Rockville, Maryland, USA, # RC208946). pcDNA3.1-NAA30-V5 was previously described and was constructed using cDNA from a human cell line ( ).

Techniques: Western Blot, Transfection, Construct, Two Tailed Test, Immunoprecipitation, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

doi: 10.1101/2022.09.01.505523

Figure Lengend Snippet: (A and B) The protein level of endogenous UBE2M in HAP1 WT and NAA30 -KO cells transfected with the indicated siRNAs for 72 h was assessed by immunoblotting. (C) Schematic representation of protein capture by peptide pulldown. A set of 11-mer peptides derived from the N-terminal sequence of UBE2M were C-terminally labelled with K-biotin ( MI KLFSLKQQK(K-biotin)) and conjugated to streptavidin magnetic beads. The first two residues were replaced to represent different N-termini (XY-UBE2M). Biotinylated UBE2M peptides were incubated with cell extracts and the pulled-down proteins were identified by immunoblot analysis. ( D) In vitro peptide pulldown assay of UBR4-V5 expressed in HeLa cells using acetylated and non-Nt-acetylated UBE2M peptide. (E) XY-UBE2M peptide pulldown assay with UBE2M peptides bearing different N-terminal amino acids and UBR4-V5 expressed in HeLa cells. (F) In vitro peptide pulldown assay of UBR4-N-FLAG expressed in HAP1 WT cells using acetylated and non-Nt-acetylated UBE2M peptide and X-nsP4 controls peptides. The UBR4-N construct contains the UBR-box (yellow), which is the substrate recognition domain of the UBR proteins. * indicates saturated UBE2A band. (G) HAP1 WT and NAA30 -KO cells were transfected with siCtrl or siUBR4 for 72 h and protein abundance was determined by TMT-based quantitative proteomics (see Table S5 ). Intensity profile plot showing protein levels of the top 100 proteins with abundance profiles most similar to UBE2M (blue trace) (FDR = 0.01, S0 = 0.1). The intensity profiles of RGS10, ARLB, CAPSN1, DIS3, and HK2 are highlighted in orange. (H) UBR4 knockdown stabilizes the protein levels of non-Nt-acetylated RGS10, HK1, DIS3, UBE2F, ARFRP1 and CAPNS1 in NAA30 -KO cells. HAP1 WT and NAA30 -KO cells were transfected with siCtrl or siUBR4 for 72 h followed by immunoblotting using the indicated antibodies.

Article Snippet: The plasmid pCMV6-UBE2M-Myc-DDK (DDK is the same as FLAG®) was obtained from OriGene Technologies (Rockville, Maryland, USA, # RC208946). pcDNA3.1-NAA30-V5 was previously described and was constructed using cDNA from a human cell line ( ).

Techniques: Transfection, Western Blot, Derivative Assay, Sequencing, Magnetic Beads, Incubation, In Vitro, Construct

Journal: bioRxiv

Article Title: N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

doi: 10.1101/2022.09.01.505523

Figure Lengend Snippet: ( Left ) The NatC complex co-translationally acetylates proteins harboring a hydrophobic residue in the second position (MΦ-). Following acetylation, the NEDD8 E2 ligases Ac-UBE2M and Ac-UBE2F promote cullin neddylation, resulting in proteasomal degradation of targeted substrates, Ac-ARFRP1 is targeted to the Golgi where it plays a role in the secretory pathway, while the hypothetical proteins Ac-X and Ac-Y are thought to affect the secretory pathway and mitochondria, respectively. ( Right ) Loss of NatC leads to proteasomal and, in some cases, lysosomal degradation of non-Nt-acetylated substrates primarily via the Arg/N-recognin UBR4-KCMF1 and to some extent via UBR1 and UBR2. Targeted degradation of unacetylated non-Nt-acetylated NatC substrates leads to decreased cullin neddylation, increased mitochondrial elongation and fragmentation, and is thought to affect intracellular trafficking.

Article Snippet: The plasmid pCMV6-UBE2M-Myc-DDK (DDK is the same as FLAG®) was obtained from OriGene Technologies (Rockville, Maryland, USA, # RC208946). pcDNA3.1-NAA30-V5 was previously described and was constructed using cDNA from a human cell line ( ).

Techniques:

Journal: bioRxiv

Article Title: N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

doi: 10.1101/2022.09.01.505523

Figure Lengend Snippet: (A) MS-spectra for the N-terminal peptide of endogenous IST1 (P53990) from HAP1 WT and NatC KO cells following trypsin digestion and strong cation exchange (SCX) enrichment. (B) Bar graph showing the degree of Nt-acetylation of IST1 in HAP1 WT and NatC KO cells as determined by proteomics. Data are shown as mean ± SD (n = 4). (C) Endogenous IST1 protein levels from indicated HAP1 cells determined by immunoblotting. (D) Immunoblot analysis of confirmed and putative NatC substrates using total cell extract from HAP1 WT and NatC KO cells. (E) N-terminal variants of UBE2M-FLAG were expressed in HAP1 NAA30 -KO cells and protein levels were determined by immunoblotting. (F) HAP1 WT and NAA30 -KO cells were transfected with the indicated UBE2M-V5-P2A-GST-GFP reporter construct, and protein levels were determined by immunoblot analysis. UBE2M-V5 levels were normalized to GST-GFP and expressed relative to WT sample. Data are shown as mean ± SD of four independent experiments. ***p < 0.0004; two-tailed unpaired t test. (G) NAA30-WT-V5 and NAA30-mut-V5 was immunoprecipitated from HeLa cell extracts and used in Nt-acetylation assays with [ 14 C]-acetyl-CoA and synthetic peptides representing the NatC substrates UBE2M (MIKL) and ARFRP1 (MYTL), and the NAA80/NatH substrate β-actin (DDDI). The experiment was performed three independent times with three technical replicates each. Data of one representative setup is shown as mean ± SD. (H) NatC regulates the protein level of UBE2M, UBE2F ARFRP1, and CAPNS1. Immunoblot analysis of HAP1 WT and NAA30 -KO cells transfected with control V5 plasmid, NAA30-V5 or the catalytically dead mutant NAA30-mut-V5. (I) HAP1 WT and NAA30 -KO cells were treated with proteasomal (MG132 and bortezomib (BMZ)) and lysosomal inhibitors (bafilomycin A (BafA), leupeptin (LP) or ammonium chloride (NH4Cl)) for 6 h followed by immunoblot analysis using the indicated antibodies. DMSO served as vehicle control.

Article Snippet: The bicistronic pUC-UBE2M-V5-P2A-GST-GFP plasmid was custom-made by VectorBuilder (Chicago, Illinois, USA) and expresses the indicated UBE2M P2A construct from a CMV promoter. pcDNA6.2-hUBR4-V5-Lumio plasmid was a kind gift from Dr. Yong Tae Kwon, Seoul National University College of Medicine, and contains a 15.9-kb human UBR4 open reading frame ( ).

Techniques: Western Blot, Transfection, Construct, Two Tailed Test, Immunoprecipitation, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

doi: 10.1101/2022.09.01.505523

Figure Lengend Snippet: (A and B) The protein level of endogenous UBE2M in HAP1 WT and NAA30 -KO cells transfected with the indicated siRNAs for 72 h was assessed by immunoblotting. (C) Schematic representation of protein capture by peptide pulldown. A set of 11-mer peptides derived from the N-terminal sequence of UBE2M were C-terminally labelled with K-biotin ( MI KLFSLKQQK(K-biotin)) and conjugated to streptavidin magnetic beads. The first two residues were replaced to represent different N-termini (XY-UBE2M). Biotinylated UBE2M peptides were incubated with cell extracts and the pulled-down proteins were identified by immunoblot analysis. ( D) In vitro peptide pulldown assay of UBR4-V5 expressed in HeLa cells using acetylated and non-Nt-acetylated UBE2M peptide. (E) XY-UBE2M peptide pulldown assay with UBE2M peptides bearing different N-terminal amino acids and UBR4-V5 expressed in HeLa cells. (F) In vitro peptide pulldown assay of UBR4-N-FLAG expressed in HAP1 WT cells using acetylated and non-Nt-acetylated UBE2M peptide and X-nsP4 controls peptides. The UBR4-N construct contains the UBR-box (yellow), which is the substrate recognition domain of the UBR proteins. * indicates saturated UBE2A band. (G) HAP1 WT and NAA30 -KO cells were transfected with siCtrl or siUBR4 for 72 h and protein abundance was determined by TMT-based quantitative proteomics (see Table S5 ). Intensity profile plot showing protein levels of the top 100 proteins with abundance profiles most similar to UBE2M (blue trace) (FDR = 0.01, S0 = 0.1). The intensity profiles of RGS10, ARLB, CAPSN1, DIS3, and HK2 are highlighted in orange. (H) UBR4 knockdown stabilizes the protein levels of non-Nt-acetylated RGS10, HK1, DIS3, UBE2F, ARFRP1 and CAPNS1 in NAA30 -KO cells. HAP1 WT and NAA30 -KO cells were transfected with siCtrl or siUBR4 for 72 h followed by immunoblotting using the indicated antibodies.

Article Snippet: The bicistronic pUC-UBE2M-V5-P2A-GST-GFP plasmid was custom-made by VectorBuilder (Chicago, Illinois, USA) and expresses the indicated UBE2M P2A construct from a CMV promoter. pcDNA6.2-hUBR4-V5-Lumio plasmid was a kind gift from Dr. Yong Tae Kwon, Seoul National University College of Medicine, and contains a 15.9-kb human UBR4 open reading frame ( ).

Techniques: Transfection, Western Blot, Derivative Assay, Sequencing, Magnetic Beads, Incubation, In Vitro, Construct

Journal: bioRxiv

Article Title: N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

doi: 10.1101/2022.09.01.505523

Figure Lengend Snippet: ( Left ) The NatC complex co-translationally acetylates proteins harboring a hydrophobic residue in the second position (MΦ-). Following acetylation, the NEDD8 E2 ligases Ac-UBE2M and Ac-UBE2F promote cullin neddylation, resulting in proteasomal degradation of targeted substrates, Ac-ARFRP1 is targeted to the Golgi where it plays a role in the secretory pathway, while the hypothetical proteins Ac-X and Ac-Y are thought to affect the secretory pathway and mitochondria, respectively. ( Right ) Loss of NatC leads to proteasomal and, in some cases, lysosomal degradation of non-Nt-acetylated substrates primarily via the Arg/N-recognin UBR4-KCMF1 and to some extent via UBR1 and UBR2. Targeted degradation of unacetylated non-Nt-acetylated NatC substrates leads to decreased cullin neddylation, increased mitochondrial elongation and fragmentation, and is thought to affect intracellular trafficking.

Article Snippet: The bicistronic pUC-UBE2M-V5-P2A-GST-GFP plasmid was custom-made by VectorBuilder (Chicago, Illinois, USA) and expresses the indicated UBE2M P2A construct from a CMV promoter. pcDNA6.2-hUBR4-V5-Lumio plasmid was a kind gift from Dr. Yong Tae Kwon, Seoul National University College of Medicine, and contains a 15.9-kb human UBR4 open reading frame ( ).

Techniques:

Journal: International Journal of Medical Sciences

Article Title: Inhibition of CRL-NEDD8 pathway as a new approach to enhance ATRA-induced differentiation of acute promyelocytic leukemia cells

doi: 10.7150/ijms.23782

Figure Lengend Snippet: ATRA inhibits cullin1- and cullin3-mediated CRL-E3 ligases and results in substrate protein accumulation in NB4 cells. Effect on CRL components and substrates after treatment of NB4 cells with 1μM ATRA for 0, 24, 48 and 72 hours. (A) Immunoblotting for cullin1, cullin3 and Rig-G. (B) For two cullin1 F-box proteins, Skp2 and βTrCP, and the cullin3 adaptor protein KLHL20. (C) For the SCFskp2 substrate p27 kip , SCFβTrCP substrate DEPTOR and cullin3-CRL substrate DAPK1. (D) For E1-activating enzyme NAE1 and E2-conjugating enzyme UBE2M. The expression of β-actin was used as loading control.

Article Snippet: The following primary antibodies were used in this study: rabbit polyclonal anti- Rig-G antibody was described previously ; anti-Cul 1 was obtained from Invitrogen (Grand Island, NY); anti-Cul 3 was purchased from BD (Franklin Lakes, NJ); anti-DAPK1 was produced by Sigma (St. Louis, MO); antibodies against LC3, NAE1, p27 kip , p-Beclin1 and βTrCP were from Cell Signaling Technologies Inc. (Beverly, MA); anti-NEDD8 was purchased from Abcam (Cambridge, UK); anti-UBE2M and β-actin were from ABclonal (USA); anti-DEPTOR and Skp2 antibodies, anti-mouse IgG, and anti-rabbit IgG were obtained from Santa Cruz Biotechnology Inc. (USA); anti-Beclin1 and KLHL20 were produced by Abgent (USA).

Techniques: Western Blot, Expressing

Journal: International Journal of Medical Sciences

Article Title: Inhibition of CRL-NEDD8 pathway as a new approach to enhance ATRA-induced differentiation of acute promyelocytic leukemia cells

doi: 10.7150/ijms.23782

Figure Lengend Snippet: Inhibition of neddylation by MLN4924 induce S phage arrest and promotes apoptosis of NB4 cells. (A) NB4 cells were treated with MLN4924 (0, 20, 40 and 80 nM) for 0, 24, 48 and 72 hours, and the protein levels of cullin1 were detected by western blotting, with β-actin used as loading control. (B) NB4 cells were exposed to MLN4924 (0, 20, 40, 80 and 160 nM), the growth curve was formed. (C) NB4 cells were treated with MLN4924 for 48 h, stained with PI, and examined with flow cytometry assays. (D) NB4 cells were treated with MLN4924 for 48 h, stained with Annexin-V-FITC and PI, and examined with flow cytometry assays.

Article Snippet: The following primary antibodies were used in this study: rabbit polyclonal anti- Rig-G antibody was described previously ; anti-Cul 1 was obtained from Invitrogen (Grand Island, NY); anti-Cul 3 was purchased from BD (Franklin Lakes, NJ); anti-DAPK1 was produced by Sigma (St. Louis, MO); antibodies against LC3, NAE1, p27 kip , p-Beclin1 and βTrCP were from Cell Signaling Technologies Inc. (Beverly, MA); anti-NEDD8 was purchased from Abcam (Cambridge, UK); anti-UBE2M and β-actin were from ABclonal (USA); anti-DEPTOR and Skp2 antibodies, anti-mouse IgG, and anti-rabbit IgG were obtained from Santa Cruz Biotechnology Inc. (USA); anti-Beclin1 and KLHL20 were produced by Abgent (USA).

Techniques: Inhibition, Western Blot, Staining, Flow Cytometry

Journal: International Journal of Medical Sciences

Article Title: Inhibition of CRL-NEDD8 pathway as a new approach to enhance ATRA-induced differentiation of acute promyelocytic leukemia cells

doi: 10.7150/ijms.23782

Figure Lengend Snippet: Inhibition of neddylation by MLN4924 induced autophagy by up-regulating DAPK1 and Beclin1. NB4 cells were treated with MLN4924 (0, 20, 40, 80 and 160 nM) for 24 h, and the levels of Nedd8, DAPK1, Beclin1, p-Beclin1 and LC3 were examined by Western blot. β-actin was used as loading control.

Article Snippet: The following primary antibodies were used in this study: rabbit polyclonal anti- Rig-G antibody was described previously ; anti-Cul 1 was obtained from Invitrogen (Grand Island, NY); anti-Cul 3 was purchased from BD (Franklin Lakes, NJ); anti-DAPK1 was produced by Sigma (St. Louis, MO); antibodies against LC3, NAE1, p27 kip , p-Beclin1 and βTrCP were from Cell Signaling Technologies Inc. (Beverly, MA); anti-NEDD8 was purchased from Abcam (Cambridge, UK); anti-UBE2M and β-actin were from ABclonal (USA); anti-DEPTOR and Skp2 antibodies, anti-mouse IgG, and anti-rabbit IgG were obtained from Santa Cruz Biotechnology Inc. (USA); anti-Beclin1 and KLHL20 were produced by Abgent (USA).

Techniques: Inhibition, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Inhibition of CRL-NEDD8 pathway as a new approach to enhance ATRA-induced differentiation of acute promyelocytic leukemia cells

doi: 10.7150/ijms.23782

Figure Lengend Snippet: Inhibition of neddylation by MLN4924 enhance ATRA induced autophagy. NB4 cells were treated with ATRA (0.01 μM) and/or MLN4924 (40 nM) for 24 h, and the expression of cullin3, cullin1, p27 kip , DAPK1, Beclin1, p-Beclin1 and LC3 were analyzed by Western blot. The expression of β-actin was used as loading control.

Article Snippet: The following primary antibodies were used in this study: rabbit polyclonal anti- Rig-G antibody was described previously ; anti-Cul 1 was obtained from Invitrogen (Grand Island, NY); anti-Cul 3 was purchased from BD (Franklin Lakes, NJ); anti-DAPK1 was produced by Sigma (St. Louis, MO); antibodies against LC3, NAE1, p27 kip , p-Beclin1 and βTrCP were from Cell Signaling Technologies Inc. (Beverly, MA); anti-NEDD8 was purchased from Abcam (Cambridge, UK); anti-UBE2M and β-actin were from ABclonal (USA); anti-DEPTOR and Skp2 antibodies, anti-mouse IgG, and anti-rabbit IgG were obtained from Santa Cruz Biotechnology Inc. (USA); anti-Beclin1 and KLHL20 were produced by Abgent (USA).

Techniques: Inhibition, Expressing, Western Blot

Journal: Cancers

Article Title: UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11

doi: 10.3390/cancers13194901

Figure Lengend Snippet: UBE2M overexpression in HCC cell lines and patient tissues, and cytotoxic and anti-proliferative effect of UBE2M depletion. ( A ) Endogenous expression level of UBE2M in hepatocellular carcinoma cell lines by Western blotting. ( B ) Effect of UBE2M depletion on the viability of HepG2, Hep3B and Huh7 cells. Cells were transfected with control and/or UBE2M siRNA-#1/siRNA-#2 and its cell viability was evaluated by MTT assay. Data represent means ± S.D from three independent experiments. ( C ) Effect of UBE2M depletion on the number of colonies in HepG2, Hep3B and Huh7 cells transfected with control and/or siRNA. Cells were cultured for 2 weeks in 12 well culture plate and stained. The number of colonies was counted. ( D ) UBE2M expression level in 80 patients’ liver cancer tissues and paired adjacent tissues using immunohistochemistry. 40× magnification. Data represent means ± S.D from three independent experiments. ** p < 0.01, *** p < 0.001 vs. untreated control. ( E ) Effect of UBE2M depletion on PARP cleavage in HepG2, Hep3B and Huh7 cells transfected with control and/or UBE2M siRNA. Cells were lysed and immunoblotted with antibodies of PARP, cleaved caspase-3, UBE2M and β-actin. ( F ) Effect of UBE2M depletion on Snail, E-cadherin in HepG2 cells.

Article Snippet: The cells were seeded onto culture plates overnight and transfected with the mixtures of p53 siRNA or UBE2M siRNA or negative control siRNA purchased from Bioneer (Daejeon, ROK) adjusted at 40 nM by using an INTERFERin transfection reagent (Polyplus, Illkirch, Illkirch, France) according to the manufacturer’s protocol.

Techniques: Over Expression, Expressing, Western Blot, Transfection, Control, MTT Assay, Cell Culture, Staining, Immunohistochemistry

Journal: Cancers

Article Title: UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11

doi: 10.3390/cancers13194901

Figure Lengend Snippet: Differentially expressed gene profile is mainly associated with p53-related signaling and UBE2M depletion induces cell-cycle arrest and apoptosis in HCCs. ( A ) Heat map of genes enriched in UBE2M-depleted HepG2 cells. Blue and red represent increased and decreased expression of genes, respectively. ( B ) Gene ontology analysis for related signaling pathways. Green and red represent increased and decreased expression of genes, respectively. ( C ) Gene analysis for TP53-, UBE2M-, RPL11-, MDM2-related genes in a pie chart. ( D ) Effect of UBE2M depletion on TP53, Bax and PUMA in HepG2 cells by qRT-PCR. RNAs isolated from HepG2 cells transfected with control and/or UBE2M siRNA were lysed and subjected to qRT-PCR. * p < 0.05, ** < 0.01, *** p < 0.001 vs. untreated control.

Article Snippet: The cells were seeded onto culture plates overnight and transfected with the mixtures of p53 siRNA or UBE2M siRNA or negative control siRNA purchased from Bioneer (Daejeon, ROK) adjusted at 40 nM by using an INTERFERin transfection reagent (Polyplus, Illkirch, Illkirch, France) according to the manufacturer’s protocol.

Techniques: Expressing, Protein-Protein interactions, Quantitative RT-PCR, Isolation, Transfection, Control

Journal: Cancers

Article Title: UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11

doi: 10.3390/cancers13194901

Figure Lengend Snippet: UBE2M depletion activates p53 and maintains its stability in HCCs. ( A ) Effect of UBE2M depletion on p53 in HepG2 cells. HepG2 cells were transfected with control siRNA and UBE2M siRNA or p53 siRNA for 72 h and were subjected to Western blotting with antibodies of p53, UBE2M and β-actin. ( B ) Effect of UBE2M depletion on p53 in Hep3B cells. Hep3B cells were transfected by control siRNA, UBE2M siRNA, pcDNA3.0 and p53 plasmids and were subjected to Western blotting with antibodies of p53, UBE2M and β-actin. ( C ) Effect of p53 depletion on UBE2M in HepG2 cells. ( D ) Effect of UBE2M depletion and/or p53 knockdown on p53 in HepG2 cells. ( E ) Effect of UBE2M overexpression on p53 in Hep3B cells. Hep3B cells were transfected with pcDNA3.0 RGS/His UBE2M and p53 plasmids and then were subjected to Western blotting with antibodies of p53, UBE2M and β-actin. ( F ) Effect of UBE2M overexpression on p53 in HepG2 cells exposed to doxorubicin. HepG2 cells were transfected with control siRNA, UBE2M siRNA, pcDNA3.0 and p53 plasmids with or without p53 activator Doxorubicin (0.1 μM) treatment and then were subjected to Western blotting with antibodies of p53, UBE2M and β-actin. ( G ) Effect of UBE2M depletion on p53 in HepG2 cells exposed to doxorubicin. ( H ) Effect of UBE2M overexpression on p53 stability in HepG2 cells in the presence of cycloheximide. HepG2 cells transfected with control siRNA and RGS/His UBE2M for 72 h were treated with cycloheximide (CHX) for 30, 60 and 90 min and were subjected to Western blotting with antibodies of p53, UBE2M and β-actin. Three independent assays were conducted in triplicate. ( I ) Effect of UBE2M depletion on p53 stability in HCT116p53+/+ cells in the presence of cycloheximide. HCT116p53+/+ cells transfected with control and UBE2M shRNA for 72 h were treated with CHX for 30, 60 and 90 min before harvesting cells and were subjected to Western blotting with antibodies of p53, UBE2M and β-actin. Three independent assays were conducted in triplicate. * p < 0.05 vs. siCTL. ** p < 0.01 vs. pcDNA3.0.

Article Snippet: The cells were seeded onto culture plates overnight and transfected with the mixtures of p53 siRNA or UBE2M siRNA or negative control siRNA purchased from Bioneer (Daejeon, ROK) adjusted at 40 nM by using an INTERFERin transfection reagent (Polyplus, Illkirch, Illkirch, France) according to the manufacturer’s protocol.

Techniques: Transfection, Control, Western Blot, Knockdown, Over Expression, shRNA

Journal: Cancers

Article Title: UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11

doi: 10.3390/cancers13194901

Figure Lengend Snippet: Ectopic expression of UBE2M enhances degradation of exogenous p53 mediated by MDM2 in HepG2 cells. ( A ) Location of UBE2M and p53 in HepG2 cells by fractionation assay. Cytosol and nuclear fractions were isolated and subjected to Western blotting with antibodies of p53, UBE2M and β-actin. ( B ) Effect of UBE2M overexpression on p53 ubiquitination in Hep3B cells. Hep3B cells were co-transfected by plasmids (pcDNA3.0, Flag-p53, MDM2, HA-Ub and RGS/His-UBE2M) and treated with 20 μM MG132 2 h before collecting protein lysates. The lysates were lysed and immunoprecipitated with anti-HA antibody and protein G-agarose beads and immunoblotted with antibodies of Flag, UBE2M and β-actin.

Article Snippet: The cells were seeded onto culture plates overnight and transfected with the mixtures of p53 siRNA or UBE2M siRNA or negative control siRNA purchased from Bioneer (Daejeon, ROK) adjusted at 40 nM by using an INTERFERin transfection reagent (Polyplus, Illkirch, Illkirch, France) according to the manufacturer’s protocol.

Techniques: Expressing, Fractionation, Isolation, Western Blot, Over Expression, Ubiquitin Proteomics, Transfection, Immunoprecipitation

Journal: Cancers

Article Title: UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11

doi: 10.3390/cancers13194901

Figure Lengend Snippet: UBE2M binds to MDM2, but regulates p53 via their crosstalk in HepG2 cells. ( A ) UBE2M binds to MDM2, but not to p53, in HepG2 cells. Immunoprecipitation was conducted in HepG2 cells to identify the endogenous interaction between UBE2M and p53, or MDM2 in the presence of MG132. ( B ) UBE2M activates MDM2 in HepG2 cells. HepG2 cells transfected with pcDNA3.0, RGS/His-UBE2M and MDM2 plasmids were lysed and immunoblotted with anti-MDM2, UBE2M and β-actin antibody. ( C ) The colocalization between MDM2 and UBE2M in HepG2 cells at endogenous level by immunofluorescence with ALEXA 488, 596 and DAPI staining. ( D ) The colocalization between MDM2 and UBE2M at exogenous level in HepG2 cells transfected with pcDNA3.0, RGS/His-UBE2M and MDM2 plasmids by immunofluorescence with ALEXA488, 596 and DAPI staining. × 200.

Article Snippet: The cells were seeded onto culture plates overnight and transfected with the mixtures of p53 siRNA or UBE2M siRNA or negative control siRNA purchased from Bioneer (Daejeon, ROK) adjusted at 40 nM by using an INTERFERin transfection reagent (Polyplus, Illkirch, Illkirch, France) according to the manufacturer’s protocol.

Techniques: Immunoprecipitation, Transfection, Immunofluorescence, Staining

Journal: Cancers

Article Title: UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11

doi: 10.3390/cancers13194901

Figure Lengend Snippet: Ribosomal protein L11 is required for p53 activation by UBE2M depletion in HepG2 cells. ( A ) UBE2M depletion activates p53 in the presence of L11. HepG2 cells were co-transfected with control siRNA, UBE2M siRNA and L11 siRNA and were subjected to Western blotting with antibodies of p53, L11, UBE2M and β actin. Total siRNA amount of its transfection mixture was adjusted to 80 nM per one well in 6-well plates. ( B ) UBE2M binds to L11 in HepG2 cells by immunoprecipitation. HepG2 transfected cells with pcDNA3.0, RGS/His-UBE2M and Flag-L11 plasmids were lysed and immunoblotted.

Article Snippet: The cells were seeded onto culture plates overnight and transfected with the mixtures of p53 siRNA or UBE2M siRNA or negative control siRNA purchased from Bioneer (Daejeon, ROK) adjusted at 40 nM by using an INTERFERin transfection reagent (Polyplus, Illkirch, Illkirch, France) according to the manufacturer’s protocol.

Techniques: Activation Assay, Transfection, Control, Western Blot, Immunoprecipitation

Journal: Cancers

Article Title: UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11

doi: 10.3390/cancers13194901

Figure Lengend Snippet: UBE2M knockdown retards the growth of HepG2 cells implanted in Balb/c. ( A ) HepG2 cells transfected with UBE2M shRNA were stabilized and selectively proliferated by puromycin; UBE2M expression level was confirmed by immunoblotting. ( B ) The cells were subcutaneously injected in the flank of Balb/c nude mouse. The tumor size was monitored and measured with the length and width of the tumor for 39 days with a caliper. ( C , D ) Thirty-nine days after injection of UBE2M shRNA transfected HepG2 cells, mice and isolated tumors were photographed. ( E ) The tumors were fixed and paraffinized for making blocks and then IHC was conducted with antibodies of p53 and UBE2M. ×40 magnification. ( F ) The schematic diagram of UBE2M signaling associated with MDM2, p53 and RPL11. *** p < 0.001 vs. HepG2 shCTL.

Article Snippet: The cells were seeded onto culture plates overnight and transfected with the mixtures of p53 siRNA or UBE2M siRNA or negative control siRNA purchased from Bioneer (Daejeon, ROK) adjusted at 40 nM by using an INTERFERin transfection reagent (Polyplus, Illkirch, Illkirch, France) according to the manufacturer’s protocol.

Techniques: Knockdown, Transfection, shRNA, Expressing, Western Blot, Injection, Isolation