A769662

Primary antibodies to β-actin (SC-47778, 1:1000), CaMKK-ß (SC-50341, 1:1000), and AMPKα1/2 (SC-25792, 1:1000) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary antibodies to p-CaMKKβ (12818S, 1:500), mTOR (2983S, 1:1000), S6 ribosomal protein (2217S, 1:1000), and p-S6 ribosomal protein (2211S, 1:1000) were purchased from Cell Signaling (Danvers, MA). Anti-p-AMPKα1/2 (11183, 1:1000) and anti-p16 (41296, 1:1000) antibodies were from Signalway (College Park, MD, USA). Anti-LC3B (NB100-2220, 1:500) and anti-p21 (NBP2-29463, 1:500) antibodies were purchased from Novus (St. Louis, MO, USA). Anti-p-mTOR (ab63552) and anti-Ki-67 (ab15580) antibodies were purchased from Abcam (Cambridge, UK). Anti-mouse (31430), anti-rabbit (31460), anti-goat (31402), and Alexa Fluor 488 (A11008) immunoglobin G secondary antibodies (1:5000) were purchased from Invitrogen (Carlsbad, CA, USA). The anti-OR2H2 primary antibody (1:1000) was purchased from Antikoerper-online.de (Aachen, Germany). L-Cis diltiazem was from Abcam (Cambridge, UK). SQ22536 and thapsigargin were purchased from Enzo Life Science (Farmingdale, NY, USA) and U73122 was from Sigma-Aldrich (St. Louis, MO, USA). Aldehyde 13-13 was obtained from Henkel (Düsseldorf, Germany). A769662, an AMPK agonist, was purchased from Cayman (Ann Arbor, MI, USA). Rapamycin was obtained from LC Laboratories (Woburn, MA, USA).

HAP1 WT or SHMT2 KO cells were seeded in 60-mm dishes and stimulated with 1 mM formate (Sigma-Aldrich), 10 μM A769662 (Cayman Chemicals) or 1 μM AICAR (Sigma-Aldrich) as indicated. Cells were washed twice in ice-cold PBS and lysed in RIPA buffer (Thermo Scientific) containing cOmplete phosphatase and protease inhibitors (Sigma-Aldrich). Equal amount of proteins were separated by electrophoresis on 3–8% 1.0 mm Tris-Acetate NuPage gels (Thermo Scientific) and transferred to nitrocellulose using an Invitrogen XCell II Blot Module. Membranes were incubated overnight at 4 °C using the following primary antibodies: ACC phospho-Ser79 (#3661), total ACC (#3676), AMPK phospho-T172 (#2531), total AMPK (#2532) (Cell Signalling Technologies). Secondary antibodies were donkey anti-mouse 800CW and goat anti-rabbit IgG (H + L) Alexa Fluor 680 (Li-COR Biosciences and Thermofisher, respectively). Immunoblots were analysed and protein densities quantified using an Odyssey CLx imager and Image Studio Lite software (Li-COR Biosciences).