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PMC12514566
LDHA-F: GGATCTCCAACATGGCAGCCTT.
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PMC12514566
LDHA-R: AGACGGCTTTCTCCCTCTTGCT.
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PMC12514566
PKM2-F: ATGGCTGACACATTCCTGGAGC.
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PMC12514566
PKM2-R: CCTTCAACGTCTCCACTGATCG.
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PMC12514566
In summary, after the designated treatments, the cells were washed twice with ice-cold PBS.
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PMC12514566
Proteins were extracted using RIPA lysis and extraction buffer (KeyGen Biotechnology, Nanjing, China).
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PMC12514566
Equal amounts of protein (30 μg per lane) were separated on an 8–10 % SDS-PAGE gel and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA).
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PMC12514566
The PVDF membrane was then incubated with primary and secondary antibodies in sequence.
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PMC12514566
The following primary antibodies were employed for Western blot: human anti-GLUT1 antibody (diluted 1:10000; cod.
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PMC12514566
ab115730; Abcam), human anti-HK2 antibody (diluted 1:1000; cod.
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PMC12514566
ab104836; Abcam), human anti-LDHA antibody (diluted 1:2000; cod.
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PMC12514566
ab52488; Abcam), human anti-PKM2 antibody (diluted 1:1000; cod.
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PMC12514566
4053T; CST), human anti-HIF-1α antibody (diluted 1:200; cod.
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PMC12514566
ab51608; Abcam), human anti-HIF-2α antibody (diluted 1:1000; cod.
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PMC12514566
ab8365; Abcam), human anti-β-Tubulin antibody (diluted 1:1000; cod.
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PMC12514566
2128S; CST).
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PMC12514566
EBV BMRF1 antibody (diluted 1:100; cod.
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PMC12514566
sc-517584; Santa Cruz), EBV Zta antibody (diluted 1:100; cod.
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PMC12514566
sc-53904; Santa Cruz).
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PMC12514566
Protein detection was carried out using enhanced chemiluminescence (Beyotime Biotechnology, Shanghai, China).
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PMC12514566
Statistical analysis was conducted with GraphPad Prism 10.1.2 software.
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PMC12514566
Each dataset reflects three independent replicates and is presented as the mean ± SD, unless specified otherwise.
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PMC12514566
Data for each group were normalized to the mean of the control group, when applicable.
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PMC12514566
Group comparisons were performed using a two-tailed Student's t-test for two groups or one-way ANOVA for multiple groups.
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PMC12514566
A significance threshold was set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
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PMC12514566
The AGS-EBV cell line was developed to retain the same genetic background as the AGS cell line, enabling more accurate comparisons between EBV-negative and EBV-positive gastric carcinoma cell lines.
[ { "end": 11, "label": "CellLine", "start": 4, "text": "AGS-EBV" }, { "end": 84, "label": "CellLine", "start": 81, "text": "AGS" } ]
PMC12514566
This was achieved by infecting the EBV-negative AGS gastric carcinoma cell line with EBV-positive Akata cells derived from Burkitt lymphoma.
[ { "end": 51, "label": "CellLine", "start": 48, "text": "AGS" }, { "end": 103, "label": "CellLine", "start": 98, "text": "Akata" } ]
PMC12514566
The presence of EBERs in AGS-EBV cells was confirmed by ISH, as shown in Fig. 1A, validating successful EBV infection.
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PMC12514566
Fig. 12-Deoxyglucose (2-DG) treatment inhibits AGS and AGS-EBV cells proliferation under normoxic and hypoxic conditions. (
[ { "end": 50, "label": "CellLine", "start": 47, "text": "AGS" }, { "end": 62, "label": "CellLine", "start": 55, "text": "AGS-EBV" } ]
PMC12514566
Representative images of H&E staining and EBERs in situ hybridization of AGS and AGS-EBV cells.
[ { "end": 76, "label": "CellLine", "start": 73, "text": "AGS" }, { "end": 88, "label": "CellLine", "start": 81, "text": "AGS-EBV" } ]
PMC12514566
H&E staining shows cell morphology, while EBER staining demonstrates the presence of EBV in AGS-EBV cells, evidenced by brown nuclear staining.
[ { "end": 99, "label": "CellLine", "start": 92, "text": "AGS-EBV" } ]
PMC12514566
Scale bar: 50 μm. (
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PMC12514566
Quantification of relative cell numbers in AGS and AGS-EBV cells under normoxic and hypoxic conditions with and without treatment with 2-DG.
[ { "end": 46, "label": "CellLine", "start": 43, "text": "AGS" }, { "end": 58, "label": "CellLine", "start": 51, "text": "AGS-EBV" } ]
PMC12514566
Data represent the mean ± SD from three independent experiments.
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PMC12514566
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.Fig.
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PMC12514566
1 2-Deoxyglucose (2-DG) treatment inhibits AGS and AGS-EBV cells proliferation under normoxic and hypoxic conditions. (
[ { "end": 46, "label": "CellLine", "start": 43, "text": "AGS" }, { "end": 58, "label": "CellLine", "start": 51, "text": "AGS-EBV" } ]
PMC12514566
Representative images of H&E staining and EBERs in situ hybridization of AGS and AGS-EBV cells.
[ { "end": 76, "label": "CellLine", "start": 73, "text": "AGS" }, { "end": 88, "label": "CellLine", "start": 81, "text": "AGS-EBV" } ]
PMC12514566
H&E staining shows cell morphology, while EBER staining demonstrates the presence of EBV in AGS-EBV cells, evidenced by brown nuclear staining.
[ { "end": 99, "label": "CellLine", "start": 92, "text": "AGS-EBV" } ]
PMC12514566
Scale bar: 50 μm. (
[]
PMC12514566
Quantification of relative cell numbers in AGS and AGS-EBV cells under normoxic and hypoxic conditions with and without treatment with 2-DG.
[ { "end": 46, "label": "CellLine", "start": 43, "text": "AGS" }, { "end": 58, "label": "CellLine", "start": 51, "text": "AGS-EBV" } ]
PMC12514566
Data represent the mean ± SD from three independent experiments.
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PMC12514566
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
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PMC12514566
To evaluate the effect of the glycolysis inhibitor 2-deoxyglucose (2-DG) on EBVnGC and EBVaGC, AGS and AGS-EBV cells were treated with 0 mM or 5 mM 2-DG under normoxic and hypoxic conditions for 48 h. Live cell counts were collected, normalized to the normoxia AGS group, and expressed as relative cell numbers.
[ { "end": 82, "label": "CellLine", "start": 76, "text": "EBVnGC" }, { "end": 93, "label": "CellLine", "start": 87, "text": "EBVaGC" }, { "end": 98, "label": "CellLine", "start": 95, "text": "AGS" }, { "end": 110, "label": "CellLine", "start": 103, "text": "AGS-EBV" }, { "end": 264, "label": "CellLine", "start": 261, "text": "AGS" } ]
PMC12514566
Across all conditions, treatment with 2-DG significantly reduced relative cell numbers compared to controls (Fig. 1B).
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PMC12514566
Under normoxic conditions, AGS-EBV cells exhibited a higher relative cell number than AGS cells, indicating that EBV infection promotes cell proliferation under normoxia (Fig. 1B).
[ { "end": 34, "label": "CellLine", "start": 27, "text": "AGS-EBV" }, { "end": 89, "label": "CellLine", "start": 86, "text": "AGS" } ]
PMC12514566
Hypoxia alone reduced AGS-EBV cell numbers compared to normoxic conditions, while the combination of hypoxia and 2-DG further suppressed AGS-EBV cell proliferation (Fig. 1B).
[ { "end": 29, "label": "CellLine", "start": 22, "text": "AGS-EBV" }, { "end": 144, "label": "CellLine", "start": 137, "text": "AGS-EBV" } ]
PMC12514566
These findings suggest that 2-DG effectively inhibits the proliferation of both AGS and AGS-EBV cells, with AGS-EBV cells showing greater sensitivity to 2-DG, particularly under hypoxic conditions.
[ { "end": 83, "label": "CellLine", "start": 80, "text": "AGS" }, { "end": 95, "label": "CellLine", "start": 88, "text": "AGS-EBV" }, { "end": 115, "label": "CellLine", "start": 108, "text": "AGS-EBV" } ]
PMC12514566
Compared to AGS cells, AGS-EBV cells exhibited increased sensitivity to 2-DG treatment, particularly under hypoxic conditions.
[ { "end": 15, "label": "CellLine", "start": 12, "text": "AGS" }, { "end": 30, "label": "CellLine", "start": 23, "text": "AGS-EBV" } ]
PMC12514566
We hypothesized that this heightened sensitivity arises from the interplay between EBV infection, hypoxia, and cellular glucose metabolism.
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PMC12514566
To investigate this, AGS and AGS-EBV cells were cultured under normoxic and hypoxic conditions, and the expression of key glycolysis-related genes (GLUT1, HK2, LDHA, and PKM2) was assessed.
[ { "end": 24, "label": "CellLine", "start": 21, "text": "AGS" }, { "end": 36, "label": "CellLine", "start": 29, "text": "AGS-EBV" } ]
PMC12514566
Hypoxia significantly increased both mRNA and protein expression of glycolysis-related genes in both AGS and AGS-EBV cells (Fig. 2A–E).
[ { "end": 104, "label": "CellLine", "start": 101, "text": "AGS" }, { "end": 116, "label": "CellLine", "start": 109, "text": "AGS-EBV" } ]
PMC12514566
Under normoxic conditions, EBV infection markedly enhanced both mRNA (GLUT1, HK2, LDHA) and protein (GLUT1, HK2, LDHA, PKM2) expression levels in AGS-EBV cells (Fig. 2A–E).
[ { "end": 153, "label": "CellLine", "start": 146, "text": "AGS-EBV" } ]
PMC12514566
Under hypoxic conditions, EBV infection significantly upregulated the expression of glycolysis-related genes (Fig. 2A–E).
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PMC12514566
These results indicate that hypoxia induces the expression of glycolysis-associated genes, and EBV infection further amplifies this response in EBVaGC.Fig.
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PMC12514566
2EBV infection upregulates the expression of glycolytic enzymes in AGS-EBV cells under hypoxic and normoxic conditions. (
[ { "end": 74, "label": "CellLine", "start": 67, "text": "AGS-EBV" } ]
PMC12514566
A–D) Glycolytic gene expression levels were measured by RT-qPCR in AGS and AGS-EBV cells under hypoxic and normoxic conditions. (
[ { "end": 70, "label": "CellLine", "start": 67, "text": "AGS" }, { "end": 82, "label": "CellLine", "start": 75, "text": "AGS-EBV" } ]
PMC12514566
Western blot analysis of GLUT1, HK2, LDHA, and PKM2 protein levels in AGS and AGS-EBV cells under hypoxic and normoxic conditions.
[ { "end": 73, "label": "CellLine", "start": 70, "text": "AGS" }, { "end": 85, "label": "CellLine", "start": 78, "text": "AGS-EBV" } ]
PMC12514566
Data represent the mean ± SD from three independent experiments.
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PMC12514566
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.Fig.
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PMC12514566
2 EBV infection upregulates the expression of glycolytic enzymes in AGS-EBV cells under hypoxic and normoxic conditions. (
[ { "end": 75, "label": "CellLine", "start": 68, "text": "AGS-EBV" } ]
PMC12514566
A–D) Glycolytic gene expression levels were measured by RT-qPCR in AGS and AGS-EBV cells under hypoxic and normoxic conditions. (
[ { "end": 70, "label": "CellLine", "start": 67, "text": "AGS" }, { "end": 82, "label": "CellLine", "start": 75, "text": "AGS-EBV" } ]
PMC12514566
Western blot analysis of GLUT1, HK2, LDHA, and PKM2 protein levels in AGS and AGS-EBV cells under hypoxic and normoxic conditions.
[ { "end": 73, "label": "CellLine", "start": 70, "text": "AGS" }, { "end": 85, "label": "CellLine", "start": 78, "text": "AGS-EBV" } ]
PMC12514566
Data represent the mean ± SD from three independent experiments.
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PMC12514566
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
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PMC12514566
Given the pivotal roles of HIF-1α and HIF-2α in cellular responses to hypoxia, we investigated the impact of EBV infection on their expression.
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PMC12514566
AGS and AGS-EBV cells were cultured under normoxic and hypoxic conditions for 48 h, followed by total protein extraction.
[ { "end": 3, "label": "CellLine", "start": 0, "text": "AGS" }, { "end": 15, "label": "CellLine", "start": 8, "text": "AGS-EBV" } ]
PMC12514566
Western blot analysis confirmed that hypoxia induced a marked increase in HIF-1α and HIF-2α expression in both AGS and AGS-EBV cells (Fig. 3A).
[ { "end": 114, "label": "CellLine", "start": 111, "text": "AGS" }, { "end": 126, "label": "CellLine", "start": 119, "text": "AGS-EBV" } ]
PMC12514566
Notably, EBV infection further upregulated HIF-1α and HIF-2α expression under both normoxic and hypoxic conditions, with the most significant enhancement observed under hypoxia (Fig. 3A).
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PMC12514566
Nuclear-cytoplasmic fractionation experiments revealed that the EBV-induced upregulation of HIF-1α and HIF-2α predominantly occurred in the nucleus (Fig. 3B).
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PMC12514566
These findings indicate that EBV infection enhances the nuclear localization and expression of HIF-1α and HIF-2α in gastric cancer cells, with this effect being particularly pronounced under hypoxic conditions.
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PMC12514566
Fig. 3EBV infection enhances the expression HIF-1α and HIF-2α in AGS-EBV cells under hypoxic and normoxic conditions. (
[ { "end": 72, "label": "CellLine", "start": 65, "text": "AGS-EBV" } ]
PMC12514566
Western blot analysis showing the protein levels of HIF-1α and HIF-2α in AGS and AGS-EBV cells under hypoxic and normoxic conditions. (
[ { "end": 76, "label": "CellLine", "start": 73, "text": "AGS" }, { "end": 88, "label": "CellLine", "start": 81, "text": "AGS-EBV" } ]
PMC12514566
Subcellular localization of HIF-1α and HIF-2α in AGS and AGS-EBV cells under hypoxic and normoxic conditions.
[ { "end": 52, "label": "CellLine", "start": 49, "text": "AGS" }, { "end": 64, "label": "CellLine", "start": 57, "text": "AGS-EBV" } ]
PMC12514566
Fig. 3 EBV infection enhances the expression HIF-1α and HIF-2α in AGS-EBV cells under hypoxic and normoxic conditions. (
[ { "end": 73, "label": "CellLine", "start": 66, "text": "AGS-EBV" } ]
PMC12514566
Western blot analysis showing the protein levels of HIF-1α and HIF-2α in AGS and AGS-EBV cells under hypoxic and normoxic conditions. (
[ { "end": 76, "label": "CellLine", "start": 73, "text": "AGS" }, { "end": 88, "label": "CellLine", "start": 81, "text": "AGS-EBV" } ]
PMC12514566
Subcellular localization of HIF-1α and HIF-2α in AGS and AGS-EBV cells under hypoxic and normoxic conditions.
[ { "end": 52, "label": "CellLine", "start": 49, "text": "AGS" }, { "end": 64, "label": "CellLine", "start": 57, "text": "AGS-EBV" } ]
PMC12514566
To investigate whether the EBV-enhanced upregulation of hypoxia-induced glycolytic gene expression is mediated by HIF-1α or HIF-2α, specific siRNAs were used to knock down the expression of each factor individually (Fig. 4).
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PMC12514566
AGS and AGS-EBV cells were treated under hypoxic conditions for 48 h, followed by Western blot analysis.
[ { "end": 3, "label": "CellLine", "start": 0, "text": "AGS" }, { "end": 15, "label": "CellLine", "start": 8, "text": "AGS-EBV" } ]
PMC12514566
The results showed that knockdown of HIF-1α significantly suppressed the EBV-mediated upregulation of glycolytic genes, while knockdown of HIF-2α had no significant effect (Fig. 4).
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PMC12514566
These findings demonstrate that the EBV-mediated amplification of hypoxia-induced glycolytic gene expression is specifically dependent on HIF-1α, highlighting its critical role in the interaction between EBV infection and hypoxia-driven metabolic pathways.
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PMC12514566
Fig. 4Knockdown HIF-1α downregulates the glycolytic enzyme expression in AGS and AGS-EBV cells under hypoxia.
[ { "end": 76, "label": "CellLine", "start": 73, "text": "AGS" }, { "end": 88, "label": "CellLine", "start": 81, "text": "AGS-EBV" } ]
PMC12514566
Western blot analysis was performed to assess the expression levels of HK2, GLUT1, LDHA, and PKM2 proteins in AGS and AGS-EBV cells transfected with siHIF1α or siHIF2α under hypoxic conditions.
[ { "end": 113, "label": "CellLine", "start": 110, "text": "AGS" }, { "end": 125, "label": "CellLine", "start": 118, "text": "AGS-EBV" } ]
PMC12514566
Fig. 4 Knockdown HIF-1α downregulates the glycolytic enzyme expression in AGS and AGS-EBV cells under hypoxia.
[ { "end": 77, "label": "CellLine", "start": 74, "text": "AGS" }, { "end": 89, "label": "CellLine", "start": 82, "text": "AGS-EBV" } ]
PMC12514566
Western blot analysis was performed to assess the expression levels of HK2, GLUT1, LDHA, and PKM2 proteins in AGS and AGS-EBV cells transfected with siHIF1α or siHIF2α under hypoxic conditions.
[ { "end": 113, "label": "CellLine", "start": 110, "text": "AGS" }, { "end": 125, "label": "CellLine", "start": 118, "text": "AGS-EBV" } ]
PMC12514566
To investigate the mechanism by which EBV infection upregulates HIF-1α expression in AGS-EBV cells, we simulated hypoxic conditions by treating cells with 200 μM cobalt chloride (CoCl2) for 24 h, followed by cycloheximide (CHX) treatment to evaluate the impact of EBV infection on the half-life of HIF-1α under hypoxia.
[ { "end": 92, "label": "CellLine", "start": 85, "text": "AGS-EBV" } ]
PMC12514566
As shown in Fig. 5A, EBV infection did not alter the half-life of HIF-1α in gastric cancer cells.
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PMC12514566
Previous studies by Tong Xiao et al. reported that EBV infection enhances HIF-1α expression at the translational level under normoxic conditions by activating the PI3K/AKT signaling pathway in nasopharyngeal carcinoma (NPC) cells.
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PMC12514566
However, EBV infection did not activate the PI3K/AKT pathway in AGS-EBV cells under hypoxia (Fig. 5B).
[ { "end": 71, "label": "CellLine", "start": 64, "text": "AGS-EBV" } ]
PMC12514566
Next, we assessed HIF-1α mRNA expression levels in AGS and AGS-EBV cells under normoxic and hypoxic conditions.
[ { "end": 54, "label": "CellLine", "start": 51, "text": "AGS" }, { "end": 66, "label": "CellLine", "start": 59, "text": "AGS-EBV" } ]
PMC12514566
The results showed that HIF-1α mRNA expression was significantly upregulated in AGS-EBV cells compared to AGS cells, with this effect being particularly pronounced under hypoxia.
[ { "end": 87, "label": "CellLine", "start": 80, "text": "AGS-EBV" }, { "end": 109, "label": "CellLine", "start": 106, "text": "AGS" } ]
PMC12514566
These findings suggest that EBV infection enhances HIF-1α expression in EBVaGC primarily at the transcriptional level, especially under hypoxic conditions.
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PMC12514566
Fig. 5EBV infection upregulates HIF-1α mRNA expression in AGS-EBV cells. (
[ { "end": 65, "label": "CellLine", "start": 58, "text": "AGS-EBV" } ]
PMC12514566
AGS and AGS-EBV cells were treated with 200 μM cobalt chloride for 24 h to mimic hypoxic conditions, followed by treatment with the protein synthesis inhibitor cycloheximide (CHX, 100 μg/ml) for 0, 30, 60, and 120 min.
[ { "end": 3, "label": "CellLine", "start": 0, "text": "AGS" }, { "end": 15, "label": "CellLine", "start": 8, "text": "AGS-EBV" } ]
PMC12514566
HIF-1α protein levels were analyzed in each group by Western blot. (
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PMC12514566
AGS and AGS-EBV cells were subjected to hypoxia for 24 h, and the expression levels of phosphorylated AKT (p-AKT) at Ser473 and Thr308 were assessed by Western blot. (
[ { "end": 3, "label": "CellLine", "start": 0, "text": "AGS" }, { "end": 15, "label": "CellLine", "start": 8, "text": "AGS-EBV" } ]
PMC12514566
AGS and AGS-EBV cells were cultured under normoxic or hypoxic conditions for 24 h, followed by RT-qPCR to measure the relative expression levels of HIF-1α.
[ { "end": 3, "label": "CellLine", "start": 0, "text": "AGS" }, { "end": 15, "label": "CellLine", "start": 8, "text": "AGS-EBV" } ]
PMC12514566
Data are presented as mean ± SD, with statistical significance indicated (∗p < 0.05, ∗∗∗∗p < 0.0001).Fig.
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PMC12514566
5 EBV infection upregulates HIF-1α mRNA expression in AGS-EBV cells. (
[ { "end": 61, "label": "CellLine", "start": 54, "text": "AGS-EBV" } ]
PMC12514566
AGS and AGS-EBV cells were treated with 200 μM cobalt chloride for 24 h to mimic hypoxic conditions, followed by treatment with the protein synthesis inhibitor cycloheximide (CHX, 100 μg/ml) for 0, 30, 60, and 120 min.
[ { "end": 3, "label": "CellLine", "start": 0, "text": "AGS" }, { "end": 15, "label": "CellLine", "start": 8, "text": "AGS-EBV" } ]
PMC12514566
HIF-1α protein levels were analyzed in each group by Western blot. (
[]