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    E-mail address: [email protected] (B. Zheng). 1 Contributed equally
    molecular mechanisms involved in the development of AKT2 in colon cancer remain unknown.
    The most prominent metabolic feature of tumors is efficient aerobic glycolysis, known as the Warburg effect. In addition, tumor aerobic glycolysis activity is closely related to the tumor growth rate and in-vasiveness [9]. Hexokinase (HK) is the first rate-limiting enzyme in the glycolytic pathway and catalyzes the production of glucose-6-phos-phate by glucose, which both produces ATP by oxidative phosphor-ylation or glycolysis, and also participates in the synthesis of important substances (e.g., nucleotides) through the pentose phosphate pathway [10]. HK contains four subtypes (i.e., HK1, HK2, HK3, and HK4), each of which has a particular tissue specificity. Among several subtypes, HK2 was found to be significantly up-regulated in various malignant tumors, including breast cancer, malignant pleural mesothelioma, myeloma, colon cancer, pancreatic cancer, and glioblastoma [11]. Re-cent studies have shown that HK2 in tumor Conessine not only mediates the Warburg effect, but also inhibits tumor cell apoptosis and regulates autophagy to promote tumor proliferation and metastasis [12]. HK2 deletion inhibits glycolysis and oxidative phosphorylation in human hepatocarcinoma and sensitizes cells to metformin [13]. Furthermore, it has been confirmed that blocking the expression of the hk2 gene and using small molecule inhibitors of HK2 can kill various tumor cells [14]. Therefore, HK2 may be a potential target for exploring tumor diagnosis and treatment.
    Several studies have also shown that HK2 may be an important downstream effector of the PI3K/AKT/mTOR signaling pathway and may contribute to the development of cancer [15]. In our previous study, we found AKT2 expression was positively correlated with HK2 expression in primary colon cancer specimens (Spearman's R = 0.711, p < .01). However, how HK2 promotes the development and pro-gression of colon cancer and whether it interacts directly with AKT2 remains poorly understood. In the present study, we aimed to elucidate how AKT2 mediates colon cancer invasion, tumorigenesis, and metas-tasis both in vitro and in vivo, and whether these effects are related to HK2.
    2. Material and methods
    Human colon cancer HCT-116 cell line (CCL-247™) and HT-29 cell line (HTB-38™) were purchased from the American Type Culture Collection (ATCC, USA). The cells were cultured in McCoy's 5a medium with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2 according to the instructions. Multiplex PCR was used to text negative for mycoplasma contamination. Nude BALB/c mice (5-weeks old) were purchased from Shanghai Slaccas Laboratory Animals Co., Ltd., China, and maintained under standard conventional condi-tions. All work performed on animals was approved by the Animal Care and Use Committee of the Zhejiang Academy of Medical Sciences.
    2.2. Immunoprecipitation of endogenous AKT2 and HK2
    Co-immunoprecipitation of proteins of interest was carried out as previously described [16]. Briefly, the supernatant containing the protein extract was incubated with protein A/G beads (Thermo, USA) overnight at 4 °C to pre-clarify the lysate bound to the A/G beads. Pre-clarified lysates were incubated with the primary antibody (Normal Rabbit IgG #2729, Akt2 Rabbit mAb #2964, Hexokinase I Rabbit mAb #2024 or Hexokinase II Rabbit mAb #2867; CST, USA) and protein A/ G beads overnight at 4 °C according to the manufacturer's instructions. The protein A/G-antibody-antigen complex was concentrated by cen-trifugation at 1000 x g for 10 min at 4 °C, followed by washing three times with PBS. The immunoprecipitated protein was then detected by electrophoresis and Western blot.  Cellular Signalling 58 (2019) 99–110
    As described previously [17], small interfering RNAs (siRNA) spe-cifically targeting AKT1, AKT2, or AKT3 were purchased from RUIBIO Co., Guangzhou, China. The siRNA sequences are presented in Table S1. The specificity of these siRNA sequences was evaluated by the online tool, BLAST, and did not display homology with other known genes. The control siRNA did not match any mammalian genes. Lipofectamine 3000 (Thermo, USA) was used to intracellularly transfect these siRNAs and silence the related genes according to the manufacturer's protocol.
    AKT2 cDNA was inserted into a pLVX-Neo vector to construct the AKT2 overexpressing vector, pLVX-Neo-AKT2. After sequencing, the target lentiviruses were achieved by transfecting either the pLVX-Neo-AKT2 or pLVX-Neo empty vector with the lentivirus packaging plasmids into 293 T cells according to the manufacturer's protocol. HCT-116 and HT-29 cells were then transfected with the resulting lentiviruses and G418 was used to screen for cell lines stably overexpressing AKT2. The cellular expression of AKT2 was confirmed by Western blot.