Lightswitch dual assay system

GSCc (15 × 10³ cells) were electroporated using the Neon® transfection system (Thermo Fisher Scientific) with either 50 ng of the control (empty) pLightSwitch_empty_Prom vector (ref #S790005) or the pLightSwitch Prom reporter plasmid for the JMY gene promoter (#S719700; SwitchGear Genomics), then immediately transferred in 96-well plates previously coated with laminin (5 µg/mL; Sigma). GSCc were irradiated (0.5 Gy) 24 h after electroporation and Luciferase reporter activity was determined at different time points using the LightSwitch Dual Assay System (SwitchGear Genomics) according to the manufacturer's instructions.

The promoter constructs of EGFR and actin (ACTB) were purchased from SwitchGear Genomics (product ID: #S714178 and #S717678). For measuring EGFR promoter activity, HEK-293T cells expressing Dox-inducible shRanBP6 were either treated with or without Dox for 72 h, and were further serum starved for 16 h. About 50 ng of actin or EGFR promoter construct and 10 ng of cypridina control were co-transfected to the cells with Fugene. The luciferase activities of renilla and cypridina were measured 48 h after transfection by following the manufacturer’s protocol (LightSwitch Dual Assay System, SwitchGear Genomics #DA010). STAT3 reporter for measuring the transcriptional activity of STAT3 was purchased from Qiagen (#CCS-9028L). For STAT3 reporter assay, both HEK293T and HEK293T-RanBP6 cell lines were treated with or without Dox for 72 h. Both of the cell lines were transfected with 100 ng of STAT3 reporter construct. The luciferase assay was developed by using Dual-Glo Luciferase Assay System from Promega (Catalog #E2920). The cells were seeded at a concentration of 15,000 cells/well in the 96-well plate, and were transfected at 60–80% confluence. Each measurement was done in biological triplicates with SpectraMax M5 multi-mode microplate readers (Molecular Devices).