1. Development and validation of soluble blockers
Development and validation of soluble blockers Retroviral construct carrying a bicistronic cassette coding for one of three signaling blockers (LAP, sEphB4, or sTie2Fc) linked to a truncated version of rabbit CD4 (tr.rbCD4), as a convenient cell surface FACS‐sortable marker, through an internal ribosomal entry site sequence (IRES). LTR = retroviral long terminal repeats.Blocker‐expressing myoblast populations, generated from control cells (Ctrl) or a clone expressing low VEGF (V‐low), were FACS‐sorted, and their purity was determined by analysis of CD4 expression (black curves) vs. isotype control (gray curves).Expression specificity was determined by RT–PCR on RNA isolated from each population, using primers specific for LAP (L), sEphB4 (E), and sTie2Fc (T), amplifying products of 781, 963, and 1,519 bp, respectively.Functional activity of the LAP blocker. HEK293N cells were transfected with a TGF‐β reporter construct, expressing luciferase under a SMAD‐dependent promoter. Conditioned medium from LAP‐expressing myoblasts (LAP) inhibited luciferase activity induced by stimulation with 0.1 and 1 ng/ml of TGF‐β1 compared to control conditioned medium from CD4 myoblasts (Ctrl). R.L.U. = relative light units. Mean ± SEM; n = 3/condition; ***P < 0.001 (one‐way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic‐transformation).Functional activity of the sTie2Fc blocker. Treatment of RAW264.7 macrophages with LPS causes upregulation of TNFα, which is inhibited by COMP‐Ang1. Real‐time qRT–PCR analysis of Tnfa gene expression shows that conditioned medium from sTie2Fc myoblasts (LPS + sTie2Fc) prevented this inhibition by 50 ng/ml COMP‐Ang1 compared to control conditioned medium from CD4 myoblasts (LPS + Ctrl). Mean ± SEM; n = 6/condition; **P < 0.01 (one‐way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic‐transformation).Functional activity of the sEphB4 blocker. Human umbilical vein endothelial cells were treated with ephrinB2‐Fc or control Fc, and phosphorylation of the EphB4 receptor was measured by ELISA. Conditioned medium from sEphB4 myoblasts (sEphB4) inhibited EphB4 phosphorylation compared to control conditioned medium from CD4 myoblasts (Ctrl). O.D. = optical density units. Mean ± SEM; n = 3/condition; *P < 0.05 (one‐way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic‐transformation).
Elena Groppa et al. EMBO Rep. 2018;embr
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