Firefly substrate

Cells were plated in 6-well plates at a density of 5 × 10⁴ cells per well and then transfected with a 8 × 3'GLI-BS luciferase construct along with the Renilla luciferase construct Pgl4 as an internal control. Cells were lysed in 100 Ml of lysis buffer (Promega, Madison, WI, USA) for 15 min at room temperature. Lysed cells were transferred into 96-well plates, and 50 μL of firefly substrate (Promega, Madison, WI, USA) was added. After measuring luciferase activity, 50 μL of Stop&Glo buffer (Promega, Madison, WI, USA) was added to the lysates. Dual luciferase activity was measured using a Glomax luminometer according to the manufacturer's instructions.

The studies were approved and processed with advice from the animal ethics board. U87MG-R-luciferase-EGFP cells with or without shSH3GLB1 were used to create a xenograft model in 6–7-week-old male NOD-SCID mice. Cells were injected into the subcutaneous flank area or brain for inoculation at numbers of 2 × 10⁶ or 1 × 10⁵, respectively. Ten days later, the animals were randomly assigned for treatment. The Luciferase activity was measured by using an IVIS imaging system (Xenogen, Alameda, CA, USA) after intraperitoneal injection of firefly substrate (80 mg/kg; Promega, Madison, WI, USA). Mice were euthanized if severe neurological or intolerable physical signs appeared. The tumor tissue was extracted for subsequent experiments. The intracranial experiments were conducted according to previous studies [5 (link)] to do survival analysis.