Fig. 1: uAUG-creating variants are associated with a decrease of ENG levels in vitro.

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^2,21,22,23 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 upstream overlapping 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, 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 monomers¹⁶. Anti-Myc and anti-ENG correspond to the used antibodies for the target protein from HeLa and HUVECs, respectively, and anti-β-actin 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. Uncropped blots are shown in Supplementary Fig. 2. All blots were processed in parallel and derive from the same 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 with standard error of the mean 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).