Lipofectamine 3000 transfection kit

The GLUT4 translocation assay was adapted from a published protocol [37 (link)]. The pEGFP-myc-GLUT4-mCherry recombinant construct for GLUT4 fusion protein was generated using the pLenti-myc-GLUT4-mCherry plasmid [37 (link)] (Addgene plasmid #64049). C2C12 cells were transfected with the recombinant plasmid using a Lipofectamine 3000 Transfection Kit (Thermo Scientific, USA). Stably transfected cells were selected and grown for further assays. C2C12 cells expressing the GLUT4 fusion protein were grown on coverslips in serum-free DMEM medium and treated with 20 μM aM1 for 24 h. The positive control group was treated with 100 nM insulin for 30 min, and the mock control was treated with PBS. After treatment, the culture medium was aspirated, and the cells were washed three times with PBS. Nonpermeabilized cells were immediately fixed with 10% formalin for 30 min, washed, and blocked with 1% BSA for 1 h at RT. After blocking, the cells were incubated with Alexa Fluor 488-conjugated c-Myc antibodies overnight at 4 °C. Subsequently, coverslips were retrieved, mounted onto slides, and visualized using a Nikon ECLIPSE Ti-S inverted microscope.

Cells were transfected using Lipofectamine 3000 transfection kit (L3000015 Thermo Fisher, Waltham, MA, USA) as per manufactures instructions with the following modification: 2.5 µg of plasmid per plate, optional p3000 reagent was utilized, transfections were incubated for 4 h. Human A549 adenocarcinoma alveolar basal epithelial cells were cultured as suggested by the ATCC. Cells were plated at 4 × 10⁵ cells/dish 24 h prior to transfection. Immediately prior to transfection cells were washed with 1 mL of PBS, and media replaced with 1 mL Opti-MEM (31985070 Thermo Fisher, Waltham, MA, USA). After 4 h, transfection media was aspirated and replaced with 2 mL Opti-MEM.