Journal: bioRxiv

Article Title: Novel uAUG creating variants in the 5’UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.12.18.520932

Figure Lengend Snippet: (A) cDNA of the long (NM_001114753.3) isoform of ENG transcripts. Positions of the identified 5’UTR uAUG-creating variants from this project and published studies – as well as position of the associated uStop codon at position c.125, and of main start (c.1) and stop (c.1975) codons are indicated. 5’UTR, 5’ Untranslated Region; CDS, CoDing Sequence; WT, wild-type; uoORF, overlapping upstream Open Reading Frame; eCDS, elongated CDS. (B) Schematic presentation of the pcDNA3.1-L-ENG constructs prepared and used for the evaluation of ENG steady-state levels in HeLa cells. Arrows indicate specific primers targeting the cDNA of ENG with extra sequences containing restriction sites to allow specific cloning in the expression vector. Position of the Myc-His tag is represented by the black square on the plasmid. CMV, cytomegalovirus promoter; WT, wild-type; Var, Variant; 5’UTR, 5’ UnTranslated Region; L, Long. (C and D) Western blot results on total proteins extracted from transfected HeLa cells with 1 µg of pcDNA3.1-L-ENG constructs or from transduced HUVEC cells with 20 MOI of lentiviruses containing ENG, respectively. Two bands of different molecular weights are observed for endoglin likely corresponding to more glycosylated (upper band) and less/non glycosylated (lower band) ENG monomers . Anti-Myc and anti-ENG correspond to the used antibodies for the target protein from HeLa and HUVECs, respectively, and anti-βactin corresponds to the antibody used against the reference protein. kDa, kilodalton; M, protein ladder; WT, wild-type, C-, negative control corresponding to pcDNA3.1-empty vector. Shown results are representative of 5 independent experiments. (E) Decrease of luciferase activity observed with uAUG-creating variants in ENG . Schematic presentation of the pGL3b-(ENG)-luciferase prepared and used in this assay is shown in the upper panel. Arrows indicate specific primers targeting the promoter of ENG with extra sequences containing restriction sites to allow specific cloning in the expression vector. Luc, luciferase. In the lower panel, shown results correspond to Firefly/Renilla ratios normalized to the wild-type (WT) in 5 independent experiments. ***, p-value < 10 −3 (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).

Article Snippet: Four hours after transfection, cells were stimulated overnight with 5 pg/ml of BMP9 in serum-free medium (R&D Systems) and luciferase activity was measured with the twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (PerkinElmer).

Techniques: Sequencing, Construct, Cloning, Expressing, Plasmid Preparation, Variant Assay, Western Blot, Transfection, Negative Control, Luciferase, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Novel uAUG creating variants in the 5’UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.12.18.520932

Figure Lengend Snippet: (A) Schematic presentation of the co-transfected constructs in this assay. BRE, BMP-response element; CMV, cytomegalovirus promoter; WT, wild-type; Var, variant; 5’UTR, 5’ UnTranslated Region; L, Long. (B) Decrease of BRE activity observed with all the analyzed variants in co-transfected NIH3T3 cells stimulated with BMP9 (5 pg/ml). Shown results correspond to the quantification of Firefly/Renilla and normalized to the wild-type (WT) (n = 4). ***, p-value < 10 −3 ; *, p-value < 5.10 −2 , ns, non-significant (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).

Article Snippet: Four hours after transfection, cells were stimulated overnight with 5 pg/ml of BMP9 in serum-free medium (R&D Systems) and luciferase activity was measured with the twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (PerkinElmer).

Techniques: Transfection, Construct, Variant Assay, Activity Assay, Comparison

Journal: Antibody Therapeutics

Article Title: Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

doi: 10.1093/abt/tbab022

Figure Lengend Snippet: The Y150-dependence of gene activation of Jurkat cells: in presence of Jurkat-CD3-NFAT-RE-Luc cells only (in Green), or in presence of Jurkat-CD38 − -CD3-NFAT-RE-Luc cells only (Jurkat-4-3-F11, in Brown), or in presence of both Jurkat-CD3-NFAT-RE-Luc cells and NCI-H929 cells (in Red), or in presence of both Jurkat- CD38 − -CD3-NFAT-RE-Luc cells and NCI-H929 cells (in Blue). In the reporter gene assay, Jurkat cells (effector cells) and NCI-H929 (target cells) were in a ratio of 1.5:1 (E:T).

Article Snippet: Promega Corporation developed a cell-based luciferase reporter gene assay to monitor the Jurkat T cell activation through binding to CD3 on the Jurkat T cells engineered with an nuclear factor of activated T cells response element (NFAT-RE) driving luciferase expression [ , ].

Techniques: Activation Assay, Reporter Gene Assay

Journal: Antibody Therapeutics

Article Title: Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

doi: 10.1093/abt/tbab022

Figure Lengend Snippet: Validation of reporter gene assay. (A) Specificity assessment of the assay. Y150 or a nonspecific antibody was incubated with both NCI-H929 cells and Jurkat T cell line 4-3-F11, and the RLU signals were measured. Y150 sample stressed under oxidization with 4% hydrogen peroxide was tested and compared with Y150 reference sample. (B) Analysis of linearity of the assay was conducted by comparing the measured potency and expected potency. Each point represents the mean of six independent experiments.

Article Snippet: Promega Corporation developed a cell-based luciferase reporter gene assay to monitor the Jurkat T cell activation through binding to CD3 on the Jurkat T cells engineered with an nuclear factor of activated T cells response element (NFAT-RE) driving luciferase expression [ , ].

Techniques: Reporter Gene Assay, Incubation

Journal: Antibody Therapeutics

Article Title: Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

doi: 10.1093/abt/tbab022

Figure Lengend Snippet: The correlations of reporter gene assay results with those from cytotoxicity assay and sandwich ELISA assay

Article Snippet: Promega Corporation developed a cell-based luciferase reporter gene assay to monitor the Jurkat T cell activation through binding to CD3 on the Jurkat T cells engineered with an nuclear factor of activated T cells response element (NFAT-RE) driving luciferase expression [ , ].

Techniques: Reporter Gene Assay, Cytotoxicity Assay, Sandwich ELISA