Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 affected the expression of epithelial–mesenchymal transition (EMT)-related factors. (A) Endogenous UBR5 knockdown changed the expression of Snail and EMT marker genes in colorectal cancer cells. Cells were collected and subjected to immunoblotting analysis and quantitative reverse transcription PCR analysis for indicated epithelial and mesenchymal markers. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (B) Immunofluorescence analysis of Snail and E-cadherin protein expression in control and shUBR5 of HCT116 cells (Snail, green; E-cadherin, red; DAPI, blue). Scale bar: 50 μm. (C) Depletion of UBR5 induced the EMT phenotype in colorectal cancer cells. Morphology of HCT116 cells after transfection with lentiviral shRNAs targeting either control or UBR5. Scale bar: 100 μm. (D) Reduction of UBR5 enhanced cell migration in vitro . Wound-healing experiments were performed to analyze changes in the migratory capacity of HCT116 control and shUBR5 cells. The histogram shows the quantitation of the relative degree of healing ( n = 3). Scale bars: 100 μm. (E) Depletion of UBR5 facilitated cell invasiveness in vitro . Transwell assay was used to analyze changes in the invasive capacity of HCT116 control and shUBR5 cells. Scale bar: 100 μm. The number of cells crossing the basement membrane was counted. The histogram shows the quantitation of the relative numbers of cells that invaded and migrated through the matrix layer ( n = 3). Scale bars: 100 μm. (F) Knockdown of UBR5 increased tumor volumes and weights. The photographs show the excised tumors from HCT116 control (left) and HCT116 shUBR5 (right) models ( n = 3). The tumor sizes (tumor volumes and weights) were subjected to comparison. ∗ P < 0.05 and ∗∗∗ P < 0.001. (G) The knockdown of UBR5 promoted tumor cell infiltration. The effect on the xenograft model in HCT116 control and shUBR5 cells was assessed by hematoxylin-eosin staining. Scale bars: 50 μm.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Expressing, Knockdown, Marker, Western Blot, Reverse Transcription, Immunofluorescence, Control, Transfection, Migration, In Vitro, Quantitation Assay, Transwell Assay, Membrane, Comparison, Staining

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: C2768S mutation abolished the effects of UBR5 on the migration and invasion of HCT116 cells. (A) C2768S mutation changed the expression of epithelial–mesenchymal transition marker genes. HCT116 cells were transfected with UBR5-Myc and UBR5 C2768S-Myc constructs. Cells were collected and subjected to immunoblotting analysis and quantitative reverse transcription PCR analysis for indicated epithelial and mesenchymal markers. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (B) Immunofluorescence analysis of Snail and E-cadherin protein expression in Mock, UBR5-Myc, and UBR5 C2768S-Myc in HCT116 cells (Snail, green; E-cadherin, red; DAPI, blue). Scale bar: 50 μm. (C) Wound-healing assays showed the migration of HCT116 cells transfected with Mock, UBR5-Myc, or UBR5 C2768S-Myc. Representative images of healing degrees at 0 and 48 h after performing the wound are shown. The histogram shows the quantitation of the relative healing degrees ( n = 3). Scale bars: 100 μm. (D) Transwell assays showed the invasiveness of HCT116 cells transfected with Mock, UBR5-Myc, or UBR5 C2768S-Myc. Representative images of the staining of the cells that invaded and migrated through the matrix layer are shown. The histogram shows the quantitation of the relative numbers of cells that invaded and migrated through the matrix layer ( n = 3). Scale bars: 100 μm. (E) Wild-type UBR5 tumors were smaller in volume and weight than the HCT116 Mock and C2768S mutant groups. The photographs show the excised tumors from HCT116 Mock, UBR5, and UBR5 C2768S models ( n = 4). The tumor sizes (tumor volumes and weights) were subjected to comparison. (F) The UBR5 C2768S mutation disrupted the UBR5-Snail axis, eliminating its regulatory effect on tumor cell invasion. Hematoxylin-eosin staining of xenograft tumors derived from HCT116 Mock, UBR5-Myc, and UBR5 C2768S-Myc cells. Scale bar: 50 μm.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Mutagenesis, Migration, Expressing, Marker, Transfection, Construct, Western Blot, Reverse Transcription, Immunofluorescence, Quantitation Assay, Staining, Comparison, Derivative Assay

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: TMEM59L regulates colorectal cancer cells proliferation, migration, and invasion. (A) Western blotting confirmed expression of TMEM59L in different CRC cell lines. (B) Western blotting detects the knockdown of TMEM59L by shRNA and overexpression of TMEM59L by plasmid. (C) Downregulation of TMEM59L suppresses cell proliferation in HCT116 cells; overexpression of TMEM59L in SW480 promotes cell proliferation. (D) The function of TMEM59L on the migration and invasion ability of CRC cells was detected by Transwell assay. (E) E‐cadherin and Vimentin were evaluated through immunofluorescence staining in TMEM59L knockdown and overexpression CRC cells.

Article Snippet: Non‐specific binding sites were blocked with 3% BSA prior to overnight incubation at 4°C with primary antibodies targeting DNA damage marker p‐γ‐H2AX (ab81299, Abcam) and EMT regulators E‐cadherin (20874‐1‐AP)/Vimentin (10366‐1‐AP, Proteintech).

Techniques: Migration, Western Blot, Expressing, Knockdown, shRNA, Over Expression, Plasmid Preparation, Transwell Assay, Immunofluorescence, Staining

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: The effect of TMEM59L and PTPRN on DNA damage, apoptosis, stemness and EMT in vivo. (A) Images of Xenograft tumors from HCT116/FU cells. (B) Average tumor volumes are measured in xenograft mice every 2 days. (C) Representative images of γ‐H2AX, TUNEL, KI67, CD133, E‐cadherin and Vimentin in tumor tissues.

Article Snippet: Non‐specific binding sites were blocked with 3% BSA prior to overnight incubation at 4°C with primary antibodies targeting DNA damage marker p‐γ‐H2AX (ab81299, Abcam) and EMT regulators E‐cadherin (20874‐1‐AP)/Vimentin (10366‐1‐AP, Proteintech).

Techniques: In Vivo, TUNEL Assay