NCI-H1944 (ATCC CRL-5907), NCI-H1395 (ATCC CRL-5868), and HepG2 (ATCC HB-8065) cells were purchased from LGC Standards (ATCC). Lung cancer cell lines were cultured in RPMI-1640 (Sigma-Aldrich, cat. No. R2405) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, cat. No. F7524) and 1% penicillin-streptomycin (P/S, Sigma-Aldrich, cat. No. P4333). HepG2 cells were cultured in low glucose DMEM (Sigma-Aldrich, cat. No. D6046) containing 10% FBS and 1% P/S. The cells were tested for Mycoplasma contamination using MycoAlert detection kit purchased from BioNordika (Lonza, cat. No. LT07-118).
NCI-H1944, NCI-H1395, and HepG2 cells were expanded and reseeded into 6-well plates at seeding densities that were estimated to result in 80% confluence after 36 and 60 hours. After an initial 12-hour incubation, the standard culture media were changed to media containing an AMPK activator (250 μM A-769662, Tocris, cat. No. 3336). The cells were harvested by trypsination after 24 and 48 h, washed with PBS, and pelleted by centrifugation (300 × g, 5 min). Each of the experiments was performed in triplicates.
Introduction of STK11wt
Stable NCI-H1944 cells expressing FLAG-LKB1 (LKB1 is STK11), or vector control were generated by retroviral transduction. pBABE FLAG-LKB1 (Addgene plasmid # 8592) virus was harvested, filtered and added to polybrene treated cells. Following a 6–8 h incubation, the cell medium was changed. Stable cell lines were generated following puromycin selection. Virus was generated in HEK293T cells. Biological triplicates were performed for the experiment.
Western blot and antibodies
For the AMPK activation experiments, each of the cell pellet samples was lysed by adding 25–100 mL of lysis buffer, heating on a shaker (95 °C, 500 rpm, 5 min, Thermomixer comfort, Eppendorf), and sonicating (50% amplitude, 1 s pulse, 30 s, Bandelin Sonopuls with a microtip, Heco Laboratorieutstyr AS). The lysis buffer was prepared fresh and contained 4% (w/v) sodium dodecyl sulfate (SDS, Fluka BioChemika), 25 mM HEPES pH 7.6 (Sigma-Aldrich), and 1 mM DL-dithiothreitol (DTT). The lysates were then centrifuged at 14,000 × g for 15 min. The total protein concentration in cell lysates was measured using a standard protocol for the Bio-Rad DC protein assay kit (with bovine serum albumin (BSA, BioRad, cat. No. 500-0007) for a five-point standard curve. Lysates containing 100 mg of protein were used for western blot analysis of each of the samples, with the exception of samples derived from NCI-H1395 cells, in which case 50 mg of protein were loaded. Western blot analyses were performed according to standard suppliers’ protocols using NuPAGE 4–12% Bis-Tris gels (Invitrogen, Thermo Fisher Scientific) and iBlot 2 Dry Blotting System with PVDF iBlot Transfer Stacks (Invitrogen). Anti-FGL1 (sc-514057, 1:300 dilution), anti-β-actin (sc-47778, 1:2,000 dilution), anti-α-tubulin (sc-5286, 1:1,000 dilution), and mouse anti-rabbit IgG-HRP (sc-2357, 1:10,000 dilution) antibodies were purchased from Santa Cruz Biotechnology, anti-LKB1 (ab15095, 1:1,000 dilution) from Abcam, anti-HNF1A (D7Z2Q, 1:1,000 dilution) from Cell Signaling Technology, and sheep anti-mouse IgG-HRP (NA931V, 1:10,000 dilution) from GE Healthcare UK. All antibodies were diluted in 5% skimmed milk powder in TBS with Tween-20 buffer (TBS-T, Thermo Scientific) with dilution ratios in accordance with the suppliers’ recommendations. Clarity Western ECL substrate (Bio-Rad) and iBright CL1500 imaging system (Invitrogen) were used for imaging of the western blots. ImageJ (v. 1.53f) software was used for densitometric analysis of the western blots. Signal intensity was normalized against the β-actin or α-tubulin loading control in each lane, thereafter the signal intensity was normalized against the mean signal of experimental control (48 h) samples in each blot. Densitometric results were reported as mean ± standard deviation and compared using two-tailed Welch test. For the introduction of STK11wt experiment, the cells were lysed in β-mercaptoethanol containing sample buffer and samples were boiled for ten minutes. Cell lysates were run on home-cast gels and wet-transferred onto PVDF membrane. Membranes were incubated in blocking solution (5% dry milk containing TBS-T) for 30 min, followed by three one-minute washes in TBS-T, and incubation with primary antibody (as described above) in TBS-T supplemented with 5% BSA overnight, and washes with TBS-T. The membrane was then incubated in 5% milk containing TBS-T with the appropriate HRP-conjugated antibodies for 1 h, washed and developed using Millipore Immobilon Western Chemiluminescent HRP mix (WBKLS0500) onto autoradiography films (SLS). Antibodies specifically used in this part were anti-GAPDH (sc-25778) from Santa Cruz Biotechnology and anti-HSP90 (Cat# 610418) from BD Biosciences.