Journal: Molecular therapy. Methods & clinical development

Article Title: Design and validation of cell-based potency assays for frataxin supplementation treatments.

doi: 10.1016/j.omtm.2024.101347

Figure Lengend Snippet: Figure 3. Stable expression of exogenous human FXN in MUT MEFs (A) A schematic of the lentivirus vector pLenti hygro FXN encoding a human miniFXN gene. The miniFXN gene used in experiments contains fragments of the endogenous FXN 50UTR, promoter and intron 1, and all five exons of the gene. (B) Representative western blot demonstrating expression of exogenous FXN. The miniFXN encodes a C-terminal HA tag that results in a slightly greater molecular weight, as detected by western blot. (C) Quantitation of endogenous Fxn (*) and exogenous FXN by western blot. Gapdh was used as a normalization control. MUT+FXN designates cells expressing exogenous, human FXN. The experiment was performed three independent times. Results are plotted as mean values with standard deviations; (**p < 0.001, ***p < 0.0001).

Article Snippet: Cloning and packaging of pLenti hygro FXN-L A miniFXN construct expressing human FXN under the control of endogenous regulatory elements was cloned into the pLenti HRELuc pGK Hygro (Addgene plasmid #118706).

Techniques: Expressing, Plasmid Preparation, Western Blot, Molecular Weight, Quantitation Assay, Control

Journal: Cancer Gene Therapy

Article Title: Stable knockdown of CREB, HIF-1 and HIF-2 by replication-competent retroviruses abrogates the responses to hypoxia in hepatocellular carcinoma

doi: 10.1038/cgt.2016.68

Figure Lengend Snippet: Quantification of the efficiency of the knockdown in infected cell lines and the effect on the expression of regulated genes. Cells, HepG2 and FLC4 were infected with each of the recombinant replication-competent retroviruses (RCRs) as specified in the figure and cultured in hypoxic conditions for 24 h. Purified mRNA was quantified by real-time quantitative PCR. Levels of messenger RNA (mRNA) were normalized to mRNAs in cells infected with vACE-NT (set as 100%) ( a–c ). Proteins were quantified following western blot analysis ( g ), and band intensities were compared with those of α-tubulin and to band intensity of cells infected with vACE-NT (set as 100%) ( d–f) . Levels of all measured mRNAs ( a – c ) and proteins ( d– f ) were statistically significantly lower when compared with vACE-NT-infected cells in both cell lines ( P <0.05). HepG2 cells infected with the various RCRs were transiently transfected with either pCRE-LUC or pHRE-LUC. Luciferase activity was determined after 48 h of exposure to hypoxia relative to cells infected with vACE-NT (presented as 100%) for each transfection. CRE (dark columns) and hypoxia-response elements (HRE) (light columns) mediated LUC activity are presented in relative light units ( P <0.001) ( h ). The levels of vascular endothelial growth factor (VEGF) secreted by HepG2 cells were determined by enzyme-linked immunosorbent assays as described in the Materials and methods section. The VEGF secretion levels in hypoxia relative to normoxia were normalized to the ratio in vACE-NT-infected cells ( P <0.01) ( i ). α-Tub=α-tubulin. Bars represent mean+s.d. for each measurement.

Article Snippet: HepG2 cells were infected with vACE-HIF-1, vACE-HIF-2, vACE-CREB, vACE-X3 or vACE-NT and seeded in 6-well plates at a concentration of 500 000 cells per well for 24 h. The infected cells were co-transfected (3 μg DNA) with either the CRE-mediated luciferase ( luc ) reporter plasmid vector, pCREluc or by the HRE-controlled luc gene reporter plasmid together with 0.25 μg of an expression vector expressing the Renilla luciferase gene, phRLSV40, as a transfection control (Promega Corp.) using FuGENE HD (Promega Corp.).

Techniques: Infection, Expressing, Recombinant, Cell Culture, Purification, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Activity Assay

Journal: Cancer Gene Therapy

Article Title: Stable knockdown of CREB, HIF-1 and HIF-2 by replication-competent retroviruses abrogates the responses to hypoxia in hepatocellular carcinoma

doi: 10.1038/cgt.2016.68

Figure Lengend Snippet: Effect of combined treatment on tumor growth monitored by light emission. HepG2 cells stably expressing the luc gene and infected with the viruses specified above were implanted subcutaneous in severe combined immunodeficiency (SCID) mice. After 3 weeks, the mice were injected twice a week with doxorubicin (DOX) in the concentrations specified in the picture. Tumor growth was monitored by light emission as described in . ( a ) One mouse from each group at day 42 is depicted. A scale bar of light emission is localized on the right side of the picture. ( b ) The average (+s.d.) relative tumor growth rate between days 14 and 42 is presented on each bar.

Article Snippet: HepG2 cells were infected with vACE-HIF-1, vACE-HIF-2, vACE-CREB, vACE-X3 or vACE-NT and seeded in 6-well plates at a concentration of 500 000 cells per well for 24 h. The infected cells were co-transfected (3 μg DNA) with either the CRE-mediated luciferase ( luc ) reporter plasmid vector, pCREluc or by the HRE-controlled luc gene reporter plasmid together with 0.25 μg of an expression vector expressing the Renilla luciferase gene, phRLSV40, as a transfection control (Promega Corp.) using FuGENE HD (Promega Corp.).

Techniques: Stable Transfection, Expressing, Infection, Injection