Fig. 1

GGAA-msats allow EwS-specific and EF1-dependent gene expression. a Fluorescence microscopy images (left) and flow cytometry histograms (right) of A-673 stably transduced with GFP under the control of a minimal promoter and with or without CRISPR/Cas9-mediated knock-in of 25 GGAA-repeats (A673_GFP_25 / A673_GFP_0) in two independent single cell clones. b Luciferase reporter assays of indicated EwS and non-EwS cell lines after co-transfection with a reporter plasmid containing the indicated number of GGAA-repeats upstream of the minimal promoter YB-TATA and a constitutively expressed Renilla-encoding plasmid. Dots indicate Firefly to Renilla luminescence ratios normalized to a reporter plasmid without GGAA-repeats for 4 biologically independent experiments. Horizontal bars indicate mean and whiskers standard deviation per group. c Luciferase reporter assays of A-673/TR/shEF1 co-transfected with the same plasmids as in Fig. 1b treated with / without Dox. Dots indicate Firefly to Renilla luminescence ratios normalized to a reporter plasmid without GGAA-repeats for 4 biologically independent experiments. Horizontal bars indicate mean and whiskers standard deviation per group. d Detection of Firefly luciferase and GAPDH in protein lysates from EwS and non-EwS cell lines transduced with pLenti_25_LT_Puro by Western blot. e Bioluminescence measurements (exposure time: 2 min) of NSG mice 14 d after intraperitoneal injection of 1 × 10⁷ TU of VSV-G-pseudotyped pLenti_25_LT or pLenti_CMV_LG lentiviral particles. f Resazurin-based cell viability assay of pLenti_25_LT_Puro-transduced and selected EwS and non-EwS cell lines 72 h after GCV addition. Dots indicate relative fluorescence units normalized to vehicle control for 4 biologically independent experiments. Lines show dose-response curves with 95% confidence interval based on a three-parameter log-logistic regression model calculated for EwS or non-EwS cells respectively. g Annexin V/PI-staining of pLenti_25_LT_Puro-transduced and selected EwS and non-EwS cell lines 72 h after GCV addition. Apoptotic cells were identified as Annexin V (APC) positive cells. Dots indicate the percentage of apoptotic cells for 4 biologically independent experiments. Horizontal bars indicate mean and whiskers the standard deviation. h Tumor volumes of pLenti_25_LT_Puro pre-transduced subcutaneous xenografts. Valganciclovir (0.5 mg/ml in drinking water enriched with 5% sucrose) or sucrose (5% in drinking water) was administered orally ad libidum once the tumor had reached an average diameter of 5 mm. i Protein concentrations in conditioned medium of pLenti_25_IX_Puro-transduced cell lines measured by ELISA. Dots indicate calculated protein concentration for 4 biologically independent experiments. Horizontal bars indicate mean and whiskers the standard deviation for EwS or non-EwS cell lines. Concentrations below the range of detectability are not depicted in the graph. j Transwell Migration Assay using conditioned medium of pLenti_25_IX_Puro-transduced and wildtype (wt) cell lines. Migrated CD3⁺ T cells were identified and counted by flow cytometry after 4 h of incubation. Dots indicate the number of migrated CD3⁺ T cells normalized to that in the wt control for each cell line for 4 biologically independent experiments. Horizontal bars indicate mean and whiskers the standard deviation. P-values were determined with two-tailed Mann-Whitney test, *: p ≤ 0.05, ****: p ≤ 0.0001