The murine NIH-3T3 fibroblast cell line was obtained from the China Infrastructure of Cell Line Resources, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. NIH-3T3 cells were grown in DMEM supplemented with 10% FBS (both Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. The following antibodies were used in the present study: Anti-STYK1 (cat. no. 18028-1-AP), anti-GLUT1 (cat. no. 21829-1-AP), anti-GLUT2 (cat. no. 20436-1-AP), anti-GLUT4 (cat. no. 21048-1-AP), anti-hexokinase (HK)1 (cat. no. 19662-1-AP), anti-platelet phosphofructokinase (PFKP) (cat. no. 13389-1-AP) and anti-pyruvate kinase (PKM)1 (cat. no. 15821-1-AP), all from ProteinTech Group, Inc.; anti-GLUT3 (cat. no. ab191071) and anti-pyruvate dehydrogenase α1 (PDHA1) (cat. no. ab168379), all from Abcam, Inc.; anti-β-actin (cat. no. 4970) and anti-β-tubulin (cat. no. 2146), both from Cell Signaling Technology, Inc.; and HRP-conjugated secondary antibody (cat. no. TA130003) from OriGene Technologies, Inc. MTT, DMSO and G418 were purchased from Sigma-Aldrich; Merck KGaA.

The pcDNA3.0 and pcDNA3.0-STYK1/NOK plasmids were constructed previously (9). For construction of pSilencer-small interfering RNA (si/siRNA) GLUT3, the single-stranded oligonucleotides (5′-AGCTTAAGTAGCTAAGTCGGTTGAAACTCGAGTTTCAACCGACTTAGCTACTTG-3′ and 5′-GATCCAAGTAGCTAAGTCGGTTGAAACTCGAGTTTCAACCGACTTAGCTACTTA-3′) were annealed to double strands before being subcloned into the HindIII and BamHI sites of pSilencer 4.1-CMV neo to form the pSilencer-siGLUT3 construct. For construction of pSilencer-small interfering RNA (si/siRNA) control, the single-stranded oligonucleotides (5′-AGCTTAATGGATCAATGGCTGGTAAACTCGAGTTTACCAGCCATTGATCCATTG-3′ and 5′-GATCCAATGGATCAATGGCTGGTAAACTCGAGTTTACCAGCCATTGATCCATTA-3′) were annealed to double strands before being subcloned into the HindIII and BamHI sites of pSilencer 4.1-CMV neo to form the pSilencer-siCtrl construct. All enzymes and reagents used for plasmid construction were purchased from Ambion, Inc. For transient transfection, NIH-3T3 cells at ~80% confluence were transiently transfected with 4 µg plasmid DNA using Lipofectamine^® 2000 transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Plasmids pcDNA3.0 or pcDNA3.0-STYK1/NOK (4 µg) were transfected into NIH-3T3 cells. After 24 h of transfection, the cell culture medium was replaced with fresh medium containing 600 µg/ml G418. After 2 weeks of screening, cell colonies were picked up and expanded in a 24-well tissue culture plate. Finally, reverse transcription-quantitative PCR (RT-qPCR) and western blotting were performed to detect STYK1/NOK expression. NIH-3T3-pcDNA3.0 stable cells were represented as ‘vehicle’ and NIH-3T3-pcDNA3.0-STYK1/NOK stable cells were represented as ‘STYK1/NOK’.

Total RNA was extracted from the cultured cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). RT-PCR was performed using the PrimeScript one-step RT-PCR kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocols. The operating conditions for RT-PCR were as follows: 50°C for 35 min for reverse transcription and 94°C for 5 min for denaturation. The PCR conditions were: 94°C for 30 sec, 50°C for 30 sec and 72°C for 50 sec, repeated for 20–30 cycles; the reaction was extended at 72°C for 10 min before the reaction product was stored at 4°C. RT-qPCR was performed using the One Step SYBR PrimeScript RT-PCR kit II (Perfect Real Time; Takara Biotechnology Co., Ltd.). The operating conditions for RT-qPCR were: 42°C for 5 min and 95°C for 10 sec; 95°C for 5 sec and 60°C for 30 sec (repeated for 40 cycles). The dissociation of the reaction products was from 55°C to 95°C as the temperature increased at a rate of 0.2°C per 10 sec. The gene expression levels of β-actin were used as an internal control. The RT-qPCR results were calculated using the 2^−ΔΔCq method (22). All primers were designed using the PrimerBank online program (https://pga.mgh.harvard.edu/primerbank/). Primer sequences are listed in Table I.

Western blotting was performed as described previously (23). Cells were collected and lysed in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing 0.1 M PMSF, protease and phosphatase inhibitor cocktail for 30 min on ice. The lysates were centrifuged at 12,000 × g at 4°C for 15 min, and the supernatant was collected. Protein concentration was determined by the BCA method. Cell lysates (20 µg) were separated using a 10% gel by SDS-PAGE and transferred to a nitrocellulose membrane (Cytiva) at 200 mA for 1.5 h. The membrane was blocked with 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, probed with a primary antibody (dilution, 1:1,000) at 4°C overnight and an appropriate secondary antibody (dilution, 1:5,000) for 1 h at room temperature. Finally, the proteins were visualized using EasySee Western Blot kit (Beijing TransGen Biotech Co., Ltd., Beijing, China), and imaged and quantified using ChemiDoc MP Imaging system (Image Lab Software, version 4.1; Bio-Rad Laboratories Co., Ltd.).

For the glucose consumption assay, cells were seeded into a 24-well culture plate in DMEM supplemented with 10% FBS. After 48 h, 100 µl culture medium was obtained from each well for the determination of glucose content using a glucose assay kit (cat. no. BC2490; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. For the lactate production assay, cells were seeded into a 6-well culture plate in normal growth medium. After 48 h, 1×10⁶ cells were collected from each well for the determination of lactate content using a lactate assay kit (cat. no. BC2230; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions.

For the proliferation assay, cells were seeded into 96-well plates at a density of 5,000 cells per well. After incubation for 24, 48 and 72 h, cell proliferation was assessed by the addition of MTT at a final concentration of 0.5 mg/ml for 1–4 h. After removing the culture medium, the cells in each well were re-suspended with 150 µl DMSO and then shaken for 10 min at 37°C to fully dissolve the crystals. The reaction products were measured at an optical density of 490 nm with a spectrophotometer.

Cell migration was analyzed using 8.0-µm Transwell^® Inserts (Corning Life Sciences). Briefly, 1×10⁵ cells were seeded into the upper chamber supplemented with 100 µl serum-free medium, while 650 µl DMEM supplemented with 10% FBS was added to the lower chamber as the inducer. Following 24 h of incubation with 5% CO₂ at 37°C, non-migrated cells in the upper chamber were removed using a cotton swab, while migrated cells in the lower chamber were stained with 0.1% crystal violet for 10 min at room temperature. Finally, the cells were washed with PBS and counted under an inverted fluorescence microscope (magnification, ×200; Olympus Corporation) using the image processing program ImageJ_v1.8.0 software (National Institutes of Health). Images of five random fields of view were captured for each group for analysis.

All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software Inc.). Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was conducted to account for the comparison of two groups and multiple groups, respectively. Data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

As aforementioned, GLUTs are required for glucose to enter cells on most occasions. Class I GLUTs (GLUT1-4) were discovered first and may serve an important role in this process. Our previous study revealed that STYK1/NOK can promote glucose uptake in NIH-3T3 cells (9), which is likely due to the enhanced expression of GLUTs. To investigate the expression profile of these four GLUTs in STYK1/NOK-mediated aerobic glycolysis in the present study, NIH-3T3 cells stably expressing STYK1/NOK were first generated. Cells transfected with pcDNA3.0 or pcDNA3.0-STYK1/NOK were subsequently selected using G418.

Following successful construction, total RNA and proteins were extracted from both the NIH-3T3-pcDNA3.0 and NIH-3T3-pcDNA3.0-STYK1/NOK stable cells. Subsequently, the mRNA and protein expression levels of STYK1/NOK and GLUT1-4 were detected using RT-qPCR (Fig. 1A and B) or western blotting (Fig. 1C), respectively. Fig. 1B shows that the mRNA expression levels of GLUT1-4, particularly GLUT3, were upregulated to varying degrees. Additionally, the results of western blot analysis were consistent with those of RT-qPCR (Fig. 1C). These data indicated that GLUT3 may have a greater impact on cell biological effects driven by STYK1/NOK. In the present study, GLUT3 was selected as the target transporter for subsequent experiments.

To gain insights into the functional involvement of GLUT3 in STYK1/NOK-induced aerobic glycolysis, siCtrl or siGLUT3 was transiently transfected into NIH-3T3 stable cells. GLUT3 knockdown was assessed by RT-PCR (Fig. 2A), RT-qPCR (Fig. 2B) and western blotting (Fig. 2C). To examine the metabolic influence of the downregulation of GLUT3, the present study subsequently analyzed the glucose uptake capacity of control and GLUT3-silenced cells by measuring the content of glucose in the culture medium. Notably, although STYK1/NOK overexpression promoted glucose consumption in both groups compared with the vehicle, the increase in STYK1/NOK-mediated glucose uptake was significantly reduced in the siGLUT3 group compared with the siCtrl group (Fig. 2D). Additionally, GLUT3-silenced NIH-3T3-STYK1/NOK stable cells exhibited reduced levels of lactate compared with non-interfering NIH-3T3-STYK1/NOK stable cells (Fig. 2E). Overall, these results suggested that metabolic reprogramming induced by STYK1/NOK in NIH-3T3 cells was impaired as a consequence of GLUT3 downregulation.

For further verification, the present study analyzed the expression levels of three rate-limiting enzymes, HK, PFKP and PKM, in the glycolysis signaling pathway, as well as PDHA1, which is one of the subunits of the pyruvate dehydrogenase complex (PDC), which catalyzes the conversion of pyruvate to acetyl-CoA (24). Fig. 3A-C shows that the mRNA expression levels of the three glycolytic enzymes were all upregulated whether or not endogenous GLUT3 was knocked down, yet the degrees of upregulation of glycolytic enzymes in NIH-3T3-STYK1/NOK stable cells were significantly decreased after GLUT3 silencing. Western blot analysis revealed similar results, with the exception that the enhanced expression levels of glycolytic enzymes mediated by STYK1/NOK almost disappeared following GLUT3 knockdown (Fig. 3D).

In contrast to the clear changes in the expression levels of glycolytic enzymes, no marked difference in PDHA1 upregulation mediated by STYK1/NOK was identified between the siCtrl and siGLUT3 groups (Fig. 3E), suggesting that GLUT3 may mainly affect STYK1/NOK-controlled aerobic glycolysis, but not the mitochondrial tricarboxylic acid (TCA) cycle.

Changes in the glucose metabolism pattern may affect cell proliferation. To investigate the influence of GLUT3 downregulation on NIH-3T3 cell properties, the present study analyzed the proliferation rate of GLUT3-silenced and control NIH-3T3 stable cells using an MTT assay. Fig. 4 shows that STYK1/NOK overexpression markedly promoted cell proliferation in the siCtrl group at several different time points (24, 48 and 72 h), which was in agreement with previous studies. However, this increase in cell proliferation caused by STYK1/NOK was markedly reduced following GLUT3 knockdown.

The malignant proliferation of cells is often accompanied by an enhanced migratory ability. The present study subsequently investigated whether loss of GLUT3 might also influence the augmented cell migration caused by STYK1/NOK. A Transwell chamber-based cell migration assay was performed using NIH-3T3 stable cells. As shown in Fig. 5, GLUT3 knockdown diminished NIH-3T3-STYK1/NOK stable cell migration compared with that in the control group. Collectively, these findings revealed that GLUT3 may be crucial to STYK1/NOK-mediated malignant transformation and tumorigenesis.