Figure 1

Increased and dysregulated protein synthesis in LCLs from a patient with FXS. Newly synthesized proteins in LCLs from a healthy control (Ctr: cell line GM10851) and a patient with FXS (FXS: cell line GM03200) were fluorescently labeled with Click-iT chemistry using a bioorthogonal amino acid (azidohomoalanine) and red-fluorescent tetramethylrhodamine (TAMRA) alkyne. LCLs were counterstained with a tubulin antibody for normalization of fluorescent signal (green). (A) Specificity of the fluorescent signal as an indicator of new protein synthesis is shown by strongly reduced red signal after treatment with the protein synthesis inhibitor anisomycin, and when azidohomoalanine (AHA) was omitted (n = 5, *P = 0.034, #P = 0.028, 1-way ANOVA with Games-Howell post hoc analyses). (B) Basal protein synthesis is increased and cannot be further increased by IL-2 stimulation in LCLs from FXS patients. Both control cells as well as FXS cells were stimulated with IL-2 (100 U/mL) for 30 min before labeling. In control cells, this leads to a significant increase in protein synthesis rates, whereas in FXS cells, protein synthesis rates are significantly decreased upon IL-2 treatment [n (Ctr untreated) = 60, n (Ctr IL-2) = 58, n (FXS untreated) = 57, n (FXS IL-2) = 58; 4 independent experiments, 2-way ANOVA shows significant interaction of treatment and genotype, Bonferroni post hoc analyses, *P = 0.005, #P = 0.002, ^≠P = 0.001). Example images for each condition in (A, scale bar is 20 µm) and (B, scale bar is 10 µm) are shown above; upper panel: newly synthesized proteins (red), lower panel: overlay with tubulin staining (green). a.u., Arbitrary units. A: □, Untreated; ■, anisomycin; ▨, no AHA; B: □, untreated; ■, IL-2.