SYBR Premix Ex Taq, PrimeScript™ RT reagent kit, gDNA Eraser and TRIzol reagent were purchased from Takara Bio Inc. (Otsu, Japan). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, 3-methyladenine (3-MA), pLKO.1 vector, puromycin and mouse anti-human anti-LC3 antibody (1:500; cat. no. L7543) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chlorquine (CQ) was obtained from InvivoGen (San Diego, CA, USA), and Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Cellulose Nitrate membranes and Luminata™ Crescendo Western Horseradish Peroxidase (HRP) Substrate were purchased from Millipore (Billerica, MA, USA). The protease and phosphatase inhibitors were obtained from Roche (Basel, Switzerland). The rabbit anti-human anti-PKM2 (1:500; cat. no. D78A4), rabbit anti-human anti-cleaved caspase 3 (1:500; cat.no. 5A1E), rabbit anti-human anti-Bcl-2 (1:500; 50E3), rabbit anti-human anti-Beclin-1 (1:1,000; cat. no. D40C5) and rabbit anti-human anti-actin (1:1,000; cat. no. 4967) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). HRP-conjugated donkey anti-mouse and anti-rabbit immunoglobulin G secondary antibodies were purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA, USA). Lipofectamine 2000 and fluorescein isothiocyanate (FITC) Annexin V/Dead Cell Apoptosis kit with FITC-Annexin V and propidium iodide (PI) was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Caspase-Glo 3/7 assay was purchased from Promega Corporation (Madison, WI, USA) and a Bicinchoninic Acid (BCA) Protein Assay kit was purchased from DingGuo (Beijing, China).

The A549 cells were grown in monolayers at 37°C in 5% CO₂ and maintained in DMEM, containing 100 units/ml penicillin and 100 μg/ml streptomycin (Sangon, Shanghai, China) sulfate, supplemented with 10% FBS. Two different target sequences for human PKM2 were identified using BLOCK iT™ RNAi designer: shPKM2-1, 5′-GCTGTGGCTCTAGACACTAAA-3′ and shPKM2-2, 5′-GTTCGGAGGTTTGATGAAATC-3′. These sequences were cloned into the pLKO.1 vector (Sigma-Aldrich). The plasmids, shPKM2-1 and shPKM2-2, were confirmed by DNA sequencing by Sangon. Lentiviral particles were produced by co-transfecting the shPKM2-1 or shPKM2-2 vector with VSVG and D8.9 packaging plas-mids (Addgene, Cambridge, MA, USA) into HEK293T cells (American Type Culture Collection, Manassas, VA, USA) using Lipofectamine 2000, according to the manufacturer's instructions. The culture medium, which contained viruses, was collected 48 h post-transfection and used to infect cells. The infected A549 cells were selected in culture medium, containing 4 μg/ml puromycin (Sigma-Aldrich) for 2 weeks.

Whole-cell lysates were extracted using radioimmunoprecipitation lysis buffer, containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 2 mM MgCl₂, supplemented with protease and phosphatase inhibitors, and the protein concentration was quantified using a BCA Protein Assay kit and a Nanodrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). The protein samples (10 μg) were separated by 12% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and then transferred onto cellulose nitrate membranes, followed by incubation in 5% non-fat milk for 1 h at room temperature. The membrane was incubated in the primary antibody at 4°C overnight and subsequently incubated in the HRP-conjugated secondary antibody at room temperature for 1 h. The target proteins were detected by using Luminata™ Crescendo Western HRP Substrate and the membranes were exposed to film.

The total RNA was extracted from cells using TRIzol reagent, according to the manufacturer's instructions. Reverse transcription of 1 μg total RNA was performed with the PrimeScript™ RT reagent kit with gDNA Eraser, according to the manufacturer's instructions. RT-PCR was performed in an Eppendorf Mastercycler^® ep realplex, according to the manufacturer's instructions. The primers were as follows: Forward: 5′-GCACAGAGCCTCGCCTT-3′ and reverse: 5′-CCTTGCACATGCCGGAG-3′ for β-actin; forward: 5′-GTGCGAGCCTCAAGTCACTCCACA-3′ and reverse: 5′-TATAAGAAGCCTCCACGCTGCCCA-3′ for PKM2; forward: 5′-GGTGGACCTGGAGAAGCTG-3′ and reverse: 5′-GGCACCCACATAAATGCC-3′ for PFKL; forward: 5′-GTGCGCATGGGTATCTACG-3′ and reverse: 5′-ACTTGCAGGATGCTGGAGAC-3′ for PFKP; forward: 5′-GGCTGCGGCTGCTAACT-3′ and reverse: 5′-CAGGGCAATGTCAGACAACT-3′ for ALDOC; forward: 5′-AGGGGTGTTTTTCCGTAAGC-3′ and reverse: 5′-GTTTCAAGCCATTCCTGCAT-3′ for ACYP1; and forward: 5′-TTGGCTGGGTGAAGAATACC-3′ and reverse: 5′-CTAGGGCTTCCAACCTTGCT-3′ for ACYP2. β-actin was used as an internal control. The comparative threshold cycle (2^−ΔΔCT) method was used to quantify the mRNA expression levels of these genes.

A pyruvate kinase activity assay was performed using 2 mg cell lysates in 50 mM Tris-HCl, 100 mM KCl, 5 mM MgCl₂, 1 mM ADP, 0.5 mM PEP, 0.2 mM ADH and 8 units lactate dehydrogenase at 37°C for 30 min. The reduction in absorbance at 340 nm, attributable to the oxidation of NADH, was measured using a microplate reader (Stat Fax-2100; Awareness Technology Inc., Palm City, FL, USA) as the pyruvate kinase activity.

The cells were plated into a 96-well plate at a density of 5×10³ cells/well. The following day, the cells were treated with or without 2 mM 3-MA for 48 h. Medium, supplemented with 0.5 mg/ml MTT was added to the cells. Following culturing for 4 h, the MTT was replaced with 100 μl/well dimethyl sulfoxide. The absorbance at 490 nm was detected using a microplate reader (Awareness Technology Inc.).

The cells were pretreated with or without 2 mM 3-MA for 48 h and harvested followed by washing in cold phosphate-buffered saline. The washed cells were centrifuged at 1,000 × g and resuspended in 1X Annexin-binding buffer at a final cell density of 1×10⁶ cells/ml. A total of 100 μl of the cells in annexin-binding buffer was obtained and mixed with 5 μl FITC/Annexin V (Component A) and 1 μl of 100 μg/ml PI working solution. Following incubation at room temperature for 15 min, 400 μl of 1X Annexin-binding buffer was added and the samples were maintained on ice. Apoptotic analyses were performed by flow cytometry (BD FACSVerse; BD Biosciences, Bedford, MA, USA), measuring the fluorescence emission at 530 nm and >575 nm. Live cells exhibited only a low level of fluorescence, apoptotic cells exhibited green fluorescence and dead cells exhibited red and green fluorescence.

The cells were seeded into white-walled 96-well plates to a final cell density at 2×10³ cells/well and were pretreated with or without 2 mM 3-MA for 48 h. The activity levels of caspases 3/7 were analyzed with the Caspase-Glo 3/7 assay, according to the manufacturer's instructions. Briefly, the plates were equilibrated to room temperature. Caspase-Glo^® 3/7 reagent (100 μl) was added into each well. Following incubation at room temperature for 30 min, the luminescent signal was detected with the Fluoroskan Ascent™ Microplate Fluorometer FL (Thermo Fisher Scientific).

The data are expressed as the mean ± standard deviation. Student's t-tests were performed to determine statistical significance of differences between experimental groups, using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The A549 cells were infected with lentivirus, containing shRNAs against PKM2. The expression levels of PKM2 were analyzed by RT-qPCR and immunoblotting. Each of the shRNAs efficiently decreased the mRNA and protein expression levels of PKM2 in the A549 cells (Fig. 1).

Pyruvate kinase activity was detected using an LDH-coupled enzyme assay and the mRNA expression levels of glycolysis-associated enzymes were analyzed by RT-qPCR in A549 cells, with or without PKM2 knockdown. Downregulation of PKM2 significantly decreased the activity of pyruvate kinase and glycolysis (Fig. 2).

The quantification of cells with punctate LC3 and the expression levels of LC3II and Beclin-1 were examined by immunoblotting to assess autophagy in the A549 cells with or without PKM2 knockdown. Downregulation of PKM2 resulted in more cells with punctate LC3, which indicated that autophagy was induced. The expression levels of LC3II and Beclin-1 were upregulated in A549 cells with PKM2 knockdown and use of an autophagy inhibitor, CQ, increased the expression levels of LC3II and Beclin-1 (Fig. 3).

Cell viability was detected using an MTT assay and apoptosis was detected using Annexin V/PI staining and flow cytometry in A549 cells with or without PKM2 knock down. Downregulation of PKM2 induced apoptosis and autophagy in the A549 cells. Inhibition of PKM2 induced-autophagy increased apoptotic cell death (Fig. 4).

Inhibition of autophagy by 3-MA further increased the activity of caspase 3/7 in A549 cells with PKM2 knockdown. Downregulation of PKM2 increased caspase 3 cleavage and decreased the expression of Bcl-2. Inhibition of autophagy using 3-MA caused increased caspase 3 cleavage and markedly lower expression of Bcl-2 (Fig. 5).