Dual luciferase assay system

MYBL2-associated GGAA-microsatellites (with ~440 bp 5′ and 3′ flanking regions) from three EwS cell lines were PCR-cloned upstream of the SV40 minimal promoter into the pGL3-Fluc vector (Promega)^{16 (link)}. Primer sequences are given in Supplementary Data 11. The presence of additional variants devoid of the GGAA-microsatellite was ruled out by WGS of the parental cell lines and Sanger sequencing of the cloned fragments. A673/TR/shEF1 cells (2 × 10⁵ per well) were transfected with the Firefly pGL3-Fluc vector containing respective microsatellites and the Renilla pGL3-Rluc vector (Promega) (ratio 100:1) in a six-well plate with 1.8 ml of growth medium. Four hours after transfection, transfection medium was replaced by medium with/without DOX (1 µg ml⁻¹; Sigma-Aldrich). Cells were lysed and assayed with a dual luciferase assay system (Berthold) after 72 h. Firefly luciferase activity was normalized to that of Renilla.

NF-κB activity assay was performed using NF-κB luciferase reporter kit (BPS Biosci-ence, Cat#: 60614) according to the manufacturer’s instruction. Macrophages with or without various treatments were co-transfected with NF-κB luciferase reporter and pRL-TK renilla luciferase reporter by jetPEI-Macrophage DNA Transfection Reagent (Polyplus-transfection, Cat#: 101000043). The luciferase activity was measured using a Dual Luciferase Assay System (Berthold Technologies).

To assess the average enhancer activity of both alleles of the PRC1-associated GGAA-mSat in a given cell, 1053 bp fragments (hg19 coordinates: chr15:91,623,953–91,625,005) including PRC1-associated GGAA-mSats (hg19 coordinates: chr15:91,624,412–91,624,459) (Supplementary Fig. 2a) from three EwS cell lines (A673, EW1, TC71) were PCR-cloned upstream of the SV40 minimal promoter into the pGL3-Fluc vector (Promega, #E1761). Primer sequences are given in Supplementary Data 8. The presence of additional variants devoid of the GGAA-mSat was ruled out by whole-genome sequencing of the parental cell lines and Sanger-sequencing of the cloned fragments. A673/TR/shEF1 cells (2 × 10⁵ per well) were co-transfected with both alleles of the mSat-containing pGL3-Fluc vectors of a given cell line in equal mass and with the Renilla pGL3-Rluc vectors (Promega) (ratio 100:1) in a six-well plate with 2 ml of growth medium. Transfection medium was replaced by medium with/without Doxycycline (Dox) (1 μg/ml; Sigma-Aldrich) 4 h after transfection. After 72 h the cells were lysed and assayed with a dual luciferase assay system (Berthold). Firefly luciferase activity was normalized to Renilla luciferase activity.