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: 9664, Cell Signaling Technology), p53 (Cat. No. sc-126, Santacruz, Rio Grande, TX, USA), MDM2 (Cat. No. sc-965, Santacruz), RPL5 (Cat. No. 14568, Cell Signaling Technology), RPL11 (Cat. No. 16277-1-AP, Proteintech, Rosemeont, IL, USA), UBE2M (Cat. No. sc-390064, Santacruz) and β-actin (Cat. No. A2228, Merck KGaA) diluted in 5% BSA in TBST overnight at 4 °C, washed three times for 10 min with TBST and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology) for 2 h. Expression was visualized by using an ECL Immunoblotting detection reagent (GE Healthcare, Chicago, IL, USA).

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: 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: 9664, Cell Signaling Technology), p53 (Cat. No. sc-126, Santacruz, Rio Grande, TX, USA), MDM2 (Cat. No. sc-965, Santacruz), RPL5 (Cat. No. 14568, Cell Signaling Technology), RPL11 (Cat. No. 16277-1-AP, Proteintech, Rosemeont, IL, USA), UBE2M (Cat. No. sc-390064, Santacruz) and β-actin (Cat. No. A2228, Merck KGaA) diluted in 5% BSA in TBST overnight at 4 °C, washed three times for 10 min with TBST and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology) for 2 h. Expression was visualized by using an ECL Immunoblotting detection reagent (GE Healthcare, Chicago, IL, USA).

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: 9664, Cell Signaling Technology), p53 (Cat. No. sc-126, Santacruz, Rio Grande, TX, USA), MDM2 (Cat. No. sc-965, Santacruz), RPL5 (Cat. No. 14568, Cell Signaling Technology), RPL11 (Cat. No. 16277-1-AP, Proteintech, Rosemeont, IL, USA), UBE2M (Cat. No. sc-390064, Santacruz) and β-actin (Cat. No. A2228, Merck KGaA) diluted in 5% BSA in TBST overnight at 4 °C, washed three times for 10 min with TBST and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology) for 2 h. Expression was visualized by using an ECL Immunoblotting detection reagent (GE Healthcare, Chicago, IL, USA).

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