Varioskan lux multimode multiplate reader

Dual‐Luciferase Reporter Assay System (Promega) was used as per provided protocol. Firefly luciferase and Renila activities were quantified using a Varioskan Lux MultiMode Multiplate reader (Thermo Fisher Scientific). Firefly luciferase signals were normalized to Renilla signals. All assays were run in triplicates.

HeLa-GLUT4 cells were seeded in 96-well plates, grown to 80% confluence, and transfected the next day with 20 nM targeting or control siRNA. After 48 h, cells were transfected with the plasmid encoding secreted alkaline phosphatase. After 24 h, fresh medium was added to the culture, and 8 h later, the medium was harvested and the cells were lysed. Alkaline phosphatase activity in the harvested medium and cell lysate was assessed using a Phospha-Light System kit (Applied Biosystems), following the manufacturer’s instructions, and detected with a luminometer (Varioskan LUX multimode multiplate reader, Thermo Fisher Scientific). The alkaline phosphatase secretion index was determined by calculating the ratio of alkaline phosphatase activity detected in the medium (secreted) to total alkaline phosphatase activity in the culture (medium plus cell lysate activity).