stable knockdown cells  (MedChemExpress)

Journal: Journal of Translational Medicine

Article Title: Glutamine metabolism reprogramming promotes bladder cancer progression via PYCR1: a multi-omics and functional validation study

doi: 10.1186/s12967-025-07386-2

Figure Lengend Snippet: PYCR1 knockdown impairs proline synthesis and suppresses the PI3K/AKT/mTOR signaling pathway in BLCA cells. ( A ) Quantification of intracellular proline levels in T24 and J82 cells after PYCR1 knockdown. ( B ) Correlation analysis showing positive associations between PYCR1 expression and SLC1A5 (left) and P5CS (right) in BLCA samples. ( C ) Western blot analysis confirming that knockdown of PYCR1 reduces protein expression levels of P5CS and SLC1A5. ( D ) Heatmap of differentially expressed genes in BLCA cells following PYCR1 knockdown from RNA-seq data. ( E - F ) Pathway enrichment analyses of downregulated ( E ) and upregulated ( F ) genes after PYCR1 knockdown, indicating involvement in PI3K-AKT and immune-related signaling pathways. ( G ) Western blot analysis validating the downregulation of PI3K, AKT, and mTOR pathway components upon PYCR1 knockdown in T24 and J82 cells. ( H ) Western blot analysis of PI3K/AKT/mTOR pathway activation following PYCR1 overexpression with or without LY294002 treatment in T24 and J82 cells. PYCR1 overexpression increased phosphorylation of PI3K, AKT, and mTOR, which was reversed by the PI3K inhibitor. Vinculin served as the loading control. Data are presented as mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01; oe: Overexpression

Article Snippet: To generate stable gene knockdown cells, lentiviral vectors expressing specific short hairpin RNA (shRNA) sequences targeting the desired gene, along with corresponding negative controls, were sourced from Genechem (Shanghai, China).

Techniques: Knockdown, Expressing, Western Blot, RNA Sequencing, Protein-Protein interactions, Activation Assay, Over Expression, Phospho-proteomics, Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) Analysis of ATOX1 transcripts in HCC tissues and adjacent normal tissues was conducted using the TCGA-LIHC database. (B) The mRNA expression of ATOX1 was analyzed in six pairs of HCC and adjacent normal tissues. (C) The protein expression levels of ATOX1 were assessed in three pairs of HCC and adjacent normal tissues (‘N’ represents adjacent normal tissue and ‘C’ represents HCC tissue). (D) ATOX1 expression in tumor tissues from 50 HCC patients was analyzed by IHC staining, with images from four representative patients presented (‘N’ represents adjacent normal tissue and ‘T’ represents tumor tissue); scale bar, 100 µm; magnification, ×20. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. HCC, hepatocellular carcinoma; LIHC, liver hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; IHC, immunohistochemistry; ATOX1, antioxidant-1.

Article Snippet: To generate stable ATOX1 knockdown cell lines, short hairpin RNA (shRNA) targeting ATOX1 was synthesized and ligated into a lentiviral vector tagged with an enhanced green fluorescent protein (OBIO Technology, Shanghai, China).

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) ATOX1 overexpression was confirmed by Western blot analysis in Huh7 and HepG2 cells. (B) The viability of Huh7 and HepG2 cells overexpressing ATOX1 was evaluated using the CCK-8 assay. (C) The number of colonies in cells overexpressing ATOX1 was assessed by the colony formation assay. (D) Cell migration in Huh7 and HepG2 cells post-ATOX1 overexpression was examined using the Transwell assay. Bar graphs depict the number of cells that migrated to the lower chambers (scale bar, 100 µm; magnification, ×10). (E) The protein expression levels of E-cadherin and N-cadherin were analyzed in Huh7 and HepG2 cells transfected with control or OE-ATOX1 plasmids. The histogram shows a relative quantitative analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. HCC, hepatocellular carcinoma; CCK-8, Cell Counting Kit-8; OE-ATOX1, ATOX1 overexpression; ATOX1, antioxidant-1.

Article Snippet: To generate stable ATOX1 knockdown cell lines, short hairpin RNA (shRNA) targeting ATOX1 was synthesized and ligated into a lentiviral vector tagged with an enhanced green fluorescent protein (OBIO Technology, Shanghai, China).

Techniques: Over Expression, Western Blot, CCK-8 Assay, Colony Assay, Migration, Transwell Assay, Expressing, Transfection, Control, Cell Counting

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) ATOX1 knockdown was confirmed by Western blot analysis in Huh7 and HepG2 cells. (B and C) The proliferative capacity of Huh7 and HepG2 cells post-ATOX1 knockdown was evaluated by the CCK-8 (B) and colony formation (C) assays. (D) The Transwell assay was performed to assess the migratory ability of Huh7 and HepG2 cells with ATOX1 knockdown (scale bar, 100 µm; magnification, ×10). (E) The protein expression of E-cadherin and N-cadherin was analyzed in Huh7 and HepG2 cells transfected with siRNA-NC or siRNA-ATOX1. (F) After subcutaneous injection of Hep3B sh-NC or Hep3B sh-ATOX1 cells in BALB/c nude mice for four weeks, tumor volume and weight were measured. * p < 0.05, ** p < 0.01, *** p < 0.001. HCC, hepatocellular carcinoma; CCK-8, Cell Counting Kit-8; si-NC, small interfering RNA targeting negative control; si-ATOX1, small interfering RNA targeting ATOX1; ATOX1, antioxidant-1.

Article Snippet: To generate stable ATOX1 knockdown cell lines, short hairpin RNA (shRNA) targeting ATOX1 was synthesized and ligated into a lentiviral vector tagged with an enhanced green fluorescent protein (OBIO Technology, Shanghai, China).

Techniques: Knockdown, Western Blot, CCK-8 Assay, Transwell Assay, Expressing, Transfection, Injection, Cell Counting, Small Interfering RNA, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) Enriched pathways through KEGG analysis in the ATOX1 knockdown group. (B) Western blot analysis of phosphorylation levels of AKT and mTOR in HCC cells with ATOX1 knockdown or overexpression. (C and D) Phosphorylation levels of AKT and mTOR in Huh7 and HepG2 cells transfected with the OE-ATOX1 plasmid for 24 h before treatment with LY294002 (20 µM) for 24 h were determined by Western blot (C). The histogram shows a relative quantitative analysis (D). (E) The effect of LY294002 on ATOX1 overexpression-induced cell proliferation was assessed using the CCK-8 assay. (F and G) The effect of LY294002 on ATOX1 overexpression-induced cell migration was measured using the Transwell assay (scale bar, 100 µm; original magnification, ×10). Bar graphs depict the number of cells that migrated to the lower chambers. * p < 0.05, ** p < 0.01, *** p < 0.001. KEGG, Kyoto Encyclopedia of Genes and Genomes; HCC, hepatocellular carcinoma; CCK-8, Cell Counting Kit-8; OE-ATOX1, ATOX1 overexpression; si-NC, small interfering RNA targeting negative control; si-ATOX1, small interfering RNA targeting ATOX1; ATOX1, antioxidant-1.

Article Snippet: To generate stable ATOX1 knockdown cell lines, short hairpin RNA (shRNA) targeting ATOX1 was synthesized and ligated into a lentiviral vector tagged with an enhanced green fluorescent protein (OBIO Technology, Shanghai, China).

Techniques: Knockdown, Western Blot, Phospho-proteomics, Over Expression, Transfection, Plasmid Preparation, CCK-8 Assay, Migration, Transwell Assay, Cell Counting, Small Interfering RNA, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A and B) Differential expression of hub genes associated with the PI3K/AKT signaling pathway in groups with ATOX1 knockdown (A) and overexpression (B) revealed by heatmap analysis. (C) PPI network of ATOX1, c-Myb, and the PI3K/AKT pathway was constructed. (D and E) The mRNA (D) and protein (E) expression levels of c-Myb were analyzed in Huh7 and HepG2 cells following ATOX1 knockdown. (F) The mRNA expression of c-Myb was analyzed in HCC cells transfected with the OE-ATOX1 plasmid for 24 h before treatment with siRNA targeting c-Myb for 24 h. (G) The PI3K/AKT pathway was analyzed by Western blot in Huh7 and HepG2 cells transfected with the OE-ATOX1 plasmid for 24 h before treatment with siRNA targeting c-Myb for 24 h. ** p < 0.01, *** p < 0.001. PPI, Protein-Protein Interaction Network; HCC, hepatocellular carcinoma; OE-ATOX1, ATOX1 overexpression; si-NC, targeting negative control; si-ATOX1, small interfering RNA targeting ATOX1; si-c-Myb, small interfering RNA targeting c-Myb; ATOX1, antioxidant-1.

Article Snippet: To generate stable ATOX1 knockdown cell lines, short hairpin RNA (shRNA) targeting ATOX1 was synthesized and ligated into a lentiviral vector tagged with an enhanced green fluorescent protein (OBIO Technology, Shanghai, China).

Techniques: Quantitative Proteomics, Knockdown, Over Expression, Construct, Expressing, Transfection, Plasmid Preparation, Western Blot, Negative Control, Small Interfering RNA

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) Huh7 cells overexpressing ATOX1 were treated with 100 µM CuSO 4 for 24 h and then cultured in a medium without CuSO 4 for 24 h. The total cellular copper content was quantified using ICP-MS analysis. (B and C) ROS levels in Huh7 and HepG2 cells with ATOX1 overexpression (B) or knockdown (C) were assessed. (D and E) The expression levels of apoptosis-related proteins, including cleaved Caspase-3, cleaved Caspase-9, BAX, and Bcl-2, were analyzed via Western blot analysis in HCC cells with ATOX1 overexpression or knockdown (D). The histogram shows a relative quantitative analysis (E). (F and G) Annexin V/PI double staining was performed to assess apoptosis in HCC cells. Bar graphs illustrate the quantitative analysis. (H) The protein expression of c-Myb in Huh7 cells transfected with siRNA targeting ATOX1 before treatment with acetylcysteine for 24 h. The histogram shows a relative quantitative analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. HCC, hepatocellular carcinoma; ICP-MS, inductively coupled plasma mass spectrometry; ROS, reactive oxygen species; PI, propidium iodide; OE-ATOX1, ATOX1 overexpression; si-NC, small interfering RNA targeting negative control; si-ATOX1, small interfering RNA targeting ATOX1; ATOX1, antioxidant-1.

Article Snippet: To generate stable ATOX1 knockdown cell lines, short hairpin RNA (shRNA) targeting ATOX1 was synthesized and ligated into a lentiviral vector tagged with an enhanced green fluorescent protein (OBIO Technology, Shanghai, China).

Techniques: Cell Culture, Over Expression, Knockdown, Expressing, Western Blot, Double Staining, Transfection, Clinical Proteomics, Mass Spectrometry, Small Interfering RNA, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) The viability of HCC cells treated with DCAC50 was analyzed by the CCK-8 assay. (B) The colony-forming ability of HCC cells treated with DCAC50 was examined via the colony formation assay. (C) ROS accumulation in HCC cells treated with DCAC50 was quantified. (D) The effect of DCAC50 on cell apoptosis was analyzed by flow cytometry. Bar graphs present quantitative analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. HCC, hepatocellular carcinoma; CCK-8, Cell Counting Kit-8; ROS, reactive oxygen species; PI, propidium iodide; ATOX1, antioxidant-1.

Article Snippet: To generate stable ATOX1 knockdown cell lines, short hairpin RNA (shRNA) targeting ATOX1 was synthesized and ligated into a lentiviral vector tagged with an enhanced green fluorescent protein (OBIO Technology, Shanghai, China).

Techniques: CCK-8 Assay, Colony Assay, Flow Cytometry, Cell Counting