ubiquitin egfp  (Addgene inc)

Journal: International Journal of Molecular Medicine

Article Title: Downregulating FGGY carbohydrate kinase domain containing promotes cell senescence by activating the p53/p21 signaling pathway in colorectal cancer

doi: 10.3892/ijmm.2025.5522

Figure Lengend Snippet: FGGY expression at both mRNA and protein levels in various type of cancer tissues. FGGY expression was analyzed in (A) COAD, (C) READ, (E) PAAD, (G) ESCA, (I) STAD and (K) LIHC from the Gene Expression Profiling Interactive Analysis database. FGGY expression was analyzed in (B) COAD, (D) READ, (F) PAAD, (H) ESCA, (J) STAD and (L) LIHC from the tissue microarray. * P<0.05. COAD, colon adenocarcinoma; ESCA esophageal cancer; FGGY, FGGY carbohydrate kinase domain containing; LIHC, liver hepatocellular carcinoma; N, normal; PAAD, pancreatic cancer; READ, rectal adenocarcinoma; STAD, gastric cancer; T, tumor; TPM, transcripts per million.

Article Snippet: Lentivirus coding shRNA targeting FGGY (LV-FGGY-RNAi; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) or the control shRNA (sh-Ctrl; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) ( ) was added at a multiplicity of infection (MOI) of 10, with medium and Hitrans P infection enhancement reagent (cat. no. REVG005; Shanghai GeneChem Co., Ltd.) reaching a total volume of 500 μ l/well.

Techniques: Expressing, Gene Expression, Microarray

Journal: International Journal of Molecular Medicine

Article Title: Downregulating FGGY carbohydrate kinase domain containing promotes cell senescence by activating the p53/p21 signaling pathway in colorectal cancer

doi: 10.3892/ijmm.2025.5522

Figure Lengend Snippet: FGGY expression at both mRNA and protein levels in CRC tissues. FGGY mRNA expression in CRC tissues and N tissues from (A) TCGA. FGGY mRNA expression in CRC tissues and N tissues from Gene Expression Omnibus (B) GSE20916 and (C) GSE113513 datasets. (D) FGGY mRNA expression labels in CRC tissues (n=80) and N tissues (n=15) on the CRC cDNA chip were analyzed by quantitative PCR. GAPDH was used as an internal control. (E) FGGY protein expression in CRC T tissues and N tissues, as determined by IHC of the tissue microarray. Representative images were taken at a magnification of ×40 or ×200 (right panel); the IHC score was calculated as intensity score x percentage score (left panel). (F) Kaplan-Meier plots of survival of patients with CRC, stratified by FGGY protein expression based on tissue microarray. * P<0.05. CRC, colorectal cancer; FGGY, FGGY carbohydrate kinase domain containing; IHC, immunohistochemistry; N, non-cancerous; TCGA, The Cancer Genome Atlas; T, tumor.

Article Snippet: Lentivirus coding shRNA targeting FGGY (LV-FGGY-RNAi; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) or the control shRNA (sh-Ctrl; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) ( ) was added at a multiplicity of infection (MOI) of 10, with medium and Hitrans P infection enhancement reagent (cat. no. REVG005; Shanghai GeneChem Co., Ltd.) reaching a total volume of 500 μ l/well.

Techniques: Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Control, Microarray, Immunohistochemistry

Journal: International Journal of Molecular Medicine

Article Title: Downregulating FGGY carbohydrate kinase domain containing promotes cell senescence by activating the p53/p21 signaling pathway in colorectal cancer

doi: 10.3892/ijmm.2025.5522

Figure Lengend Snippet: Association between FGGY expression and the clinicopathological characteristics of patients with colorectal cancer in a tissue microarray.

Article Snippet: Lentivirus coding shRNA targeting FGGY (LV-FGGY-RNAi; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) or the control shRNA (sh-Ctrl; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) ( ) was added at a multiplicity of infection (MOI) of 10, with medium and Hitrans P infection enhancement reagent (cat. no. REVG005; Shanghai GeneChem Co., Ltd.) reaching a total volume of 500 μ l/well.

Techniques: Expressing, Microarray

Journal: International Journal of Molecular Medicine

Article Title: Downregulating FGGY carbohydrate kinase domain containing promotes cell senescence by activating the p53/p21 signaling pathway in colorectal cancer

doi: 10.3892/ijmm.2025.5522

Figure Lengend Snippet: FGGY knockdown inhibits the proliferation of colorectal cancer cells. (A) mRNA and (B) protein expression levels of FGGY in HCT116 and RKO cells after transduction with sh-FGGY or sh-Ctrl was assessed by quantitative PCR and western blotting. GAPDH was used as a loading control. Band intensities were semi-quantified using ImageLab software. (C) Colony formation of HCT116 and RKO cells transduced with sh-FGGY or sh-Ctrl was assessed by colony formation assay. Colony formation was normalized to the sh-Ctrl group. (D) Viability of HCT116 and RKO cells after FGGY knockdown was determined by Cell Counting Kit-8. Data were normalized to viability at day 1 and presented as fold change. (E) Percentage of HCT116 and RKO cells transduced with sh-FGGY or sh-Ctrl in G 0 /G 1 , S and G 2 /M phases, as determined by flow cytometry. Representative flow cytometry plots and the percentage of cells at each stage are shown. (F) Apoptosis of HCT116 and RKO cells was detected by Annexin V staining and flow cytometric analysis. Representative plots and percentages of apoptotic cells are shown. * P<0.05 vs. sh-Ctrl. Ctrl, control; FGGY, FGGY carbohydrate kinase domain containing; OD, optical density; sh, short hairpin.

Article Snippet: Lentivirus coding shRNA targeting FGGY (LV-FGGY-RNAi; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) or the control shRNA (sh-Ctrl; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) ( ) was added at a multiplicity of infection (MOI) of 10, with medium and Hitrans P infection enhancement reagent (cat. no. REVG005; Shanghai GeneChem Co., Ltd.) reaching a total volume of 500 μ l/well.

Techniques: Knockdown, Expressing, Transduction, Real-time Polymerase Chain Reaction, Western Blot, Control, Software, Colony Assay, Cell Counting, Flow Cytometry, Staining

Journal: International Journal of Molecular Medicine

Article Title: Downregulating FGGY carbohydrate kinase domain containing promotes cell senescence by activating the p53/p21 signaling pathway in colorectal cancer

doi: 10.3892/ijmm.2025.5522

Figure Lengend Snippet: FGGY knockdown inhibits colorectal cancer cell growth in vivo . A xenograft nude mouse model was established to investigate the tumor growth of HCT116 and RKO cells in vivo . A total of 1.0×10 6 cells transduced with lentiviruses encoding sh-Ctrl or sh-FGGY were injected subcutaneously into the forelimb axillae (sh-Ctrl, left; sh-FGGY, right) of BALB/c nude mice. (A) Tumor volumes, (B) fluorescence intensities of tumor cells, and (C) images of tumors and tumor weights were determined and recorded. * P<0.05 vs. sh-Ctrl. Ctrl, control; FGGY, FGGY carbohydrate kinase domain containing; sh, short hairpin.

Article Snippet: Lentivirus coding shRNA targeting FGGY (LV-FGGY-RNAi; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) or the control shRNA (sh-Ctrl; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) ( ) was added at a multiplicity of infection (MOI) of 10, with medium and Hitrans P infection enhancement reagent (cat. no. REVG005; Shanghai GeneChem Co., Ltd.) reaching a total volume of 500 μ l/well.

Techniques: Knockdown, In Vivo, Transduction, Injection, Fluorescence, Control

Journal: International Journal of Molecular Medicine

Article Title: Downregulating FGGY carbohydrate kinase domain containing promotes cell senescence by activating the p53/p21 signaling pathway in colorectal cancer

doi: 10.3892/ijmm.2025.5522

Figure Lengend Snippet: Cancer-associated pathways were significantly enriched among proteins dysregulated by FGGY knockdown in HCT116 cells. HCT116 cells were transduced with lentiviruses encoding sh-FGGY or sh-Ctrl, and (A-C) isobaric tags for relative and absolute quantitation and (D-F) phosphoproteomics analyses were used to identify DEPs. (A and D) Hierarchical clustering plots and (B and E) volcano plots were used to identify DEPs (fold change >1.2, P<0.05). (C and F) KEGG pathway enrichment analysis was performed to identify functionally related gene pathways. The top 30 enriched signaling pathways are shown. Ctrl, control; DEPs, differentially expressed proteins. FGGY, FGGY carbohydrate kinase domain containing; sh, short hairpin.

Article Snippet: Lentivirus coding shRNA targeting FGGY (LV-FGGY-RNAi; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) or the control shRNA (sh-Ctrl; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) ( ) was added at a multiplicity of infection (MOI) of 10, with medium and Hitrans P infection enhancement reagent (cat. no. REVG005; Shanghai GeneChem Co., Ltd.) reaching a total volume of 500 μ l/well.

Techniques: Knockdown, Transduction, Quantitation Assay, Phospho-proteomics, Protein-Protein interactions, Control

Journal: International Journal of Molecular Medicine

Article Title: Downregulating FGGY carbohydrate kinase domain containing promotes cell senescence by activating the p53/p21 signaling pathway in colorectal cancer

doi: 10.3892/ijmm.2025.5522

Figure Lengend Snippet: FGGY knockdown promotes cell senescence by activating the p53 pathway. The correlation between FGGY and TP53 expression was analyzed using (A) Gene Expression Omnibus (accession number: GSE39582) and (B) The Cancer Genome Atlas databases. (C) Protein levels of p53, p21 and PCNA in HCT116 cells after FGGY knockdown were assessed by western blotting. GAPDH was used as a loading control. Band intensities were semi-quantified using ImageLab software. * P<0.05 vs. sh-Ctrl. (D) Expression levels of p21 and p53 proteins in HCT116/p53 +/+ and HCT116/p53 −/− cells transduced with sh-FGGY or sh-Ctrl were assessed by western blotting. GAPDH was used as a loading control. Band intensities were semi-quantified using ImageLab software. (E) Viability of HCT116/p53 +/+ and HCT116/p53 −/− cells transduced with sh-FGGY or sh-Ctrl. Results were normalized to viability on day 1. (F) Senescence-associated β-gal staining in HCT116/p53 +/+ and HCT116/p53 −/− cells after FGGY knockdown. Representative images were taken at a magnification of ×200. Immunofluorescence images showing co-localization of FGGY in chromatin foci with the SAHF markers (G) H3k9me3 and (H) HP1γ in HCT116/p53 +/+ and HCT116/p53 −/− cells after FGGY knockdown. Representative images were taken at a magnification of ×400. * P<0.05 vs. sh-Ctrl HCT116/p53 +/+ cells. β-gal, β-galactosidase; Ctrl, control; FGGY, FGGY carbohydrate kinase domain containing; H3k9me3, trimethylation of H3K9; sh, short hairpin.

Article Snippet: Lentivirus coding shRNA targeting FGGY (LV-FGGY-RNAi; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) or the control shRNA (sh-Ctrl; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) ( ) was added at a multiplicity of infection (MOI) of 10, with medium and Hitrans P infection enhancement reagent (cat. no. REVG005; Shanghai GeneChem Co., Ltd.) reaching a total volume of 500 μ l/well.

Techniques: Knockdown, Expressing, Gene Expression, Western Blot, Control, Software, Transduction, Staining, Immunofluorescence

Journal: International Journal of Molecular Medicine

Article Title: Downregulating FGGY carbohydrate kinase domain containing promotes cell senescence by activating the p53/p21 signaling pathway in colorectal cancer

doi: 10.3892/ijmm.2025.5522

Figure Lengend Snippet: Knockdown of FGGY inhibits cell senescence through the p53/p21 signaling pathway in colorectal cancer. The figure was created using Figdraw ( https://www.figdraw.com/ ). FGGY, FGGY carbohydrate kinase domain containing; sh, short hairpin.

Article Snippet: Lentivirus coding shRNA targeting FGGY (LV-FGGY-RNAi; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) or the control shRNA (sh-Ctrl; hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) ( ) was added at a multiplicity of infection (MOI) of 10, with medium and Hitrans P infection enhancement reagent (cat. no. REVG005; Shanghai GeneChem Co., Ltd.) reaching a total volume of 500 μ l/well.

Techniques: Knockdown