Camkk2 sirna

Cells were transfected with 100 nM AMPK siRNA (50 nM α1(sc-270142) and 50 nM α2 (sc-155985), Santa Cruz Biotechnology, Santa Cruz, CA), LKB1 siRNA (#L-100539-02, Dharmacon, Lafayette, CO, USA), CaMKK2 siRNA (#L-097995-02, Dharmacon, Lafayette, CO, USA) or 100 nM scrambled siRNA (Ambion #AM4011, Invitrogen, Carlsbad, CA, USA) using lipofectamin RNAiMAX (Invitrogen, Carlsbad, CA) in OPTI-MEM (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. After 24 h of transfection, culture medium was replaced with 0.1% WME. Cells were cultured for additional 24 h and then, harvested for further experiments.

siRNA transfection was carried out using ON-TARGETplus SMARTpool control siRNA and CAMKK2 siRNA (Dharmacon, Lafayette, United States). AGS cells were transfected as previously described by using RNAiMAX (Invitrogen, Grand Island, NY) (Najar et al., 2021b (link)). They were plated at a density of 60,000 cells/well in six-well plates followed by transfection with 20 nM of CAMKK2 siRNA and control siRNA in Opti-MEM media (Gibco, CA, United States) using RNAiMAX. The cells were transfected with CAMKK2 siRNA and control siRNA in Opti-MEM media using Lipofectamine RNAiMAX transfection reagent. After transfection, the media was changed to complete media (DMEM +10% FBS) and the cells were allowed to grow for 48 h. Finally, the cells were harvested using lysis buffer, and lysates were used for immunoblotting experiments to check protein expression.