si ezh2 si fbxl7 Search Results


si ezh2 si fbxl7  (MedChemExpress)
96
MedChemExpress si ezh2 si fbxl7
A A volcano plot of DEGs in 28 normal samples and 35 NSCLC samples in the GSE12472 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. B A volcano plot of DEGs in 25 normal samples and 25 NSCLC samples in the GSE27262 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. C A volcano plot of DEGs in 34 normal samples and 32 NSCLC samples in the GSE101929 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. D A volcano plot of DEGs in 6 normal samples and 6 NSCLC samples in the GSE118370 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. E Venn diagram of the 16 upregulated DEGs from the four datasets. F Venn diagram of the 15 downregulated DEGs from the four datasets. G Venn diagram showing the intersection <t>(FBXL7)</t> of the DEGs from the four datasets and 501 E3 ubiquitin ligases from the iUUCD database. H Expression of FBXL7 mRNA was downregulated in NSCLC tissue samples, as analyzed using GEPIA. Red box: tumor samples, gray box: normal samples; LUSC: lung squamous cell carcinoma, LUAD: lung adenocarcinoma. I Kaplan-Meier analysis showing the positive correlation between FBXL7 expression and the overall survival rate of patients. Red: high expression of FBXL7; black: low expression of FBXL7. J Expression of FBXL7 was downregulated in NSCLC cells (as compared with normal lung epithelial cells), as determined by RT-qPCR. K Western blot analysis showing the downregulated FBXL7 protein in NSCLC cells (as compared with normal lung epithelial cells). ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with BEAS-2B cells. The cell experiment was repeated three times independently.
Si Ezh2 Si Fbxl7, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cycloheximide (chx; 20 μg/ml)  (Beyotime)
90
Beyotime cycloheximide (chx; 20 μg/ml)
A A volcano plot of DEGs in 28 normal samples and 35 NSCLC samples in the GSE12472 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. B A volcano plot of DEGs in 25 normal samples and 25 NSCLC samples in the GSE27262 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. C A volcano plot of DEGs in 34 normal samples and 32 NSCLC samples in the GSE101929 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. D A volcano plot of DEGs in 6 normal samples and 6 NSCLC samples in the GSE118370 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. E Venn diagram of the 16 upregulated DEGs from the four datasets. F Venn diagram of the 15 downregulated DEGs from the four datasets. G Venn diagram showing the intersection <t>(FBXL7)</t> of the DEGs from the four datasets and 501 E3 ubiquitin ligases from the iUUCD database. H Expression of FBXL7 mRNA was downregulated in NSCLC tissue samples, as analyzed using GEPIA. Red box: tumor samples, gray box: normal samples; LUSC: lung squamous cell carcinoma, LUAD: lung adenocarcinoma. I Kaplan-Meier analysis showing the positive correlation between FBXL7 expression and the overall survival rate of patients. Red: high expression of FBXL7; black: low expression of FBXL7. J Expression of FBXL7 was downregulated in NSCLC cells (as compared with normal lung epithelial cells), as determined by RT-qPCR. K Western blot analysis showing the downregulated FBXL7 protein in NSCLC cells (as compared with normal lung epithelial cells). ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with BEAS-2B cells. The cell experiment was repeated three times independently.
Cycloheximide (Chx; 20 μg/Ml), supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ezh2  (Cell Signaling Technology Inc)
98
Cell Signaling Technology Inc ezh2
Fig. 6 Hypoxia increases the expression of <t>EZH2,</t> which reduces the expression of FBXL7 and promotes PFKFB4 protein stability. A Western blot analysis showing elevated expression of EZH2 and HIF-1α proteins in hypoxic A549 cells. B Verification of the binding of HIF-1α to the EZH2 promoter region, as evaluated by dual-luciferase reporter assay. C Enrichment of HIF-1α in the promoter region of EZH2, as observed by ChIP assay. D mRNA expression of EZH2, FBXL7, and PFKFB4 in normoxic or hypoxic A549 cells in the presence of si-EZH2. E Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in normoxic or hypoxic A549 cells in the presence of si- EZH2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.
Ezh2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pfkfb4  (Abcam)
95
Abcam pfkfb4
A <t>PFKFB4</t> expression was elevated in NSCLC tissue samples, as analyzed by UALCAN database. LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma. B Kaplan–Meier analysis showing the negative correlation between PFKFB4 and the overall survival rate of NSCLC patients. Red: high expression of PFKFB4; black: low expression of PFKFB4. C Co-IP analysis of the interaction of endogenous FBXL7 with PFKFB4 in 293T cells. D Co-IP analysis of the interaction of exogenous FBXL7 with PFKFB4 in 293T cells. E GST-pull down assay analysis of the direct interaction between FBXL7 and PFKFB4 in 293T cells. F PFKFB4 mRNA expression in A549 cells wasn’t changed in response to oe-FBXL7, as determined by RT-qPCR. G Western blot analysis showing inhibited PFKFB4 protein expression in A549 cells in response to oe-FBXL7. H Western blot analysis showing enhanced degradation of PFKFB4 protein in 293T cells in response to oe-FBXL7 and CHX. I Ubiquitination level of PFKFB4 protein was reduced in 293T cells in the presence of si-FBXL7, as determined by IP assay. J Ubiquitination level of PFKFB4 protein was elevated in 293T cells in the presence of oe-FBXL7, as determined by IP assay. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.
Pfkfb4, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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myc  (Abcam)
98
Abcam myc
A <t>PFKFB4</t> expression was elevated in NSCLC tissue samples, as analyzed by UALCAN database. LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma. B Kaplan–Meier analysis showing the negative correlation between PFKFB4 and the overall survival rate of NSCLC patients. Red: high expression of PFKFB4; black: low expression of PFKFB4. C Co-IP analysis of the interaction of endogenous FBXL7 with PFKFB4 in 293T cells. D Co-IP analysis of the interaction of exogenous FBXL7 with PFKFB4 in 293T cells. E GST-pull down assay analysis of the direct interaction between FBXL7 and PFKFB4 in 293T cells. F PFKFB4 mRNA expression in A549 cells wasn’t changed in response to oe-FBXL7, as determined by RT-qPCR. G Western blot analysis showing inhibited PFKFB4 protein expression in A549 cells in response to oe-FBXL7. H Western blot analysis showing enhanced degradation of PFKFB4 protein in 293T cells in response to oe-FBXL7 and CHX. I Ubiquitination level of PFKFB4 protein was reduced in 293T cells in the presence of si-FBXL7, as determined by IP assay. J Ubiquitination level of PFKFB4 protein was elevated in 293T cells in the presence of oe-FBXL7, as determined by IP assay. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.
Myc, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hif 1α  (Cell Signaling Technology Inc)
99
Cell Signaling Technology Inc hif 1α
A Western blot analysis showing elevated expression of EZH2 and <t>HIF-1α</t> proteins in hypoxic A549 cells. B Verification of the binding of HIF-1α to the EZH2 promoter region, as evaluated by dual-luciferase reporter assay. C Enrichment of HIF-1α in the promoter region of EZH2, as observed by ChIP assay. D mRNA expression of EZH2, FBXL7, and PFKFB4 in normoxic or hypoxic A549 cells in the presence of si-EZH2. E Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in normoxic or hypoxic A549 cells in the presence of si-EZH2. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.
Hif 1α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


A A volcano plot of DEGs in 28 normal samples and 35 NSCLC samples in the GSE12472 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. B A volcano plot of DEGs in 25 normal samples and 25 NSCLC samples in the GSE27262 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. C A volcano plot of DEGs in 34 normal samples and 32 NSCLC samples in the GSE101929 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. D A volcano plot of DEGs in 6 normal samples and 6 NSCLC samples in the GSE118370 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. E Venn diagram of the 16 upregulated DEGs from the four datasets. F Venn diagram of the 15 downregulated DEGs from the four datasets. G Venn diagram showing the intersection (FBXL7) of the DEGs from the four datasets and 501 E3 ubiquitin ligases from the iUUCD database. H Expression of FBXL7 mRNA was downregulated in NSCLC tissue samples, as analyzed using GEPIA. Red box: tumor samples, gray box: normal samples; LUSC: lung squamous cell carcinoma, LUAD: lung adenocarcinoma. I Kaplan-Meier analysis showing the positive correlation between FBXL7 expression and the overall survival rate of patients. Red: high expression of FBXL7; black: low expression of FBXL7. J Expression of FBXL7 was downregulated in NSCLC cells (as compared with normal lung epithelial cells), as determined by RT-qPCR. K Western blot analysis showing the downregulated FBXL7 protein in NSCLC cells (as compared with normal lung epithelial cells). ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with BEAS-2B cells. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A A volcano plot of DEGs in 28 normal samples and 35 NSCLC samples in the GSE12472 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. B A volcano plot of DEGs in 25 normal samples and 25 NSCLC samples in the GSE27262 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. C A volcano plot of DEGs in 34 normal samples and 32 NSCLC samples in the GSE101929 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. D A volcano plot of DEGs in 6 normal samples and 6 NSCLC samples in the GSE118370 dataset. Red indicates upregulated DEGs, green indicates downregulated DEGs, and black indicates no difference in gene expression. E Venn diagram of the 16 upregulated DEGs from the four datasets. F Venn diagram of the 15 downregulated DEGs from the four datasets. G Venn diagram showing the intersection (FBXL7) of the DEGs from the four datasets and 501 E3 ubiquitin ligases from the iUUCD database. H Expression of FBXL7 mRNA was downregulated in NSCLC tissue samples, as analyzed using GEPIA. Red box: tumor samples, gray box: normal samples; LUSC: lung squamous cell carcinoma, LUAD: lung adenocarcinoma. I Kaplan-Meier analysis showing the positive correlation between FBXL7 expression and the overall survival rate of patients. Red: high expression of FBXL7; black: low expression of FBXL7. J Expression of FBXL7 was downregulated in NSCLC cells (as compared with normal lung epithelial cells), as determined by RT-qPCR. K Western blot analysis showing the downregulated FBXL7 protein in NSCLC cells (as compared with normal lung epithelial cells). ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with BEAS-2B cells. The cell experiment was repeated three times independently.

Article Snippet: Cells under hypoxic conditions were treated with si-EZH2 + si-FBXL7 or combined with 2-DG (16 mM, 154-17-6, MCE).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

A Expression of FBXL7 mRNA was elevated in A549 and H1650 cells transduced with oe-FBXL7, as determined by RT-qPCR. B Western blot analysis showing the elevated FBXL7 protein expression in A549 and H1650 cells transduced with oe-FBXL7. C Viability of A549 and H1650 cells was inhibited in response to oe-FBXL7, as analyzed by CCK-8 assay. D Migration and invasion of A549 and H1650 cells were inhibited in response to oe-FBXL7 analyzed by Transwell assays. E Apoptosis of A549 and H1650 cells was promoted in response to oe-FBXL7, as analyzed by flow cytometry. ** p < 0.01, *** p < 0.001, **** p < 0.0001. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A Expression of FBXL7 mRNA was elevated in A549 and H1650 cells transduced with oe-FBXL7, as determined by RT-qPCR. B Western blot analysis showing the elevated FBXL7 protein expression in A549 and H1650 cells transduced with oe-FBXL7. C Viability of A549 and H1650 cells was inhibited in response to oe-FBXL7, as analyzed by CCK-8 assay. D Migration and invasion of A549 and H1650 cells were inhibited in response to oe-FBXL7 analyzed by Transwell assays. E Apoptosis of A549 and H1650 cells was promoted in response to oe-FBXL7, as analyzed by flow cytometry. ** p < 0.01, *** p < 0.001, **** p < 0.0001. The cell experiment was repeated three times independently.

Article Snippet: Cells under hypoxic conditions were treated with si-EZH2 + si-FBXL7 or combined with 2-DG (16 mM, 154-17-6, MCE).

Techniques: Expressing, Transduction, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Migration, Flow Cytometry

A PFKFB4 expression was elevated in NSCLC tissue samples, as analyzed by UALCAN database. LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma. B Kaplan–Meier analysis showing the negative correlation between PFKFB4 and the overall survival rate of NSCLC patients. Red: high expression of PFKFB4; black: low expression of PFKFB4. C Co-IP analysis of the interaction of endogenous FBXL7 with PFKFB4 in 293T cells. D Co-IP analysis of the interaction of exogenous FBXL7 with PFKFB4 in 293T cells. E GST-pull down assay analysis of the direct interaction between FBXL7 and PFKFB4 in 293T cells. F PFKFB4 mRNA expression in A549 cells wasn’t changed in response to oe-FBXL7, as determined by RT-qPCR. G Western blot analysis showing inhibited PFKFB4 protein expression in A549 cells in response to oe-FBXL7. H Western blot analysis showing enhanced degradation of PFKFB4 protein in 293T cells in response to oe-FBXL7 and CHX. I Ubiquitination level of PFKFB4 protein was reduced in 293T cells in the presence of si-FBXL7, as determined by IP assay. J Ubiquitination level of PFKFB4 protein was elevated in 293T cells in the presence of oe-FBXL7, as determined by IP assay. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A PFKFB4 expression was elevated in NSCLC tissue samples, as analyzed by UALCAN database. LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma. B Kaplan–Meier analysis showing the negative correlation between PFKFB4 and the overall survival rate of NSCLC patients. Red: high expression of PFKFB4; black: low expression of PFKFB4. C Co-IP analysis of the interaction of endogenous FBXL7 with PFKFB4 in 293T cells. D Co-IP analysis of the interaction of exogenous FBXL7 with PFKFB4 in 293T cells. E GST-pull down assay analysis of the direct interaction between FBXL7 and PFKFB4 in 293T cells. F PFKFB4 mRNA expression in A549 cells wasn’t changed in response to oe-FBXL7, as determined by RT-qPCR. G Western blot analysis showing inhibited PFKFB4 protein expression in A549 cells in response to oe-FBXL7. H Western blot analysis showing enhanced degradation of PFKFB4 protein in 293T cells in response to oe-FBXL7 and CHX. I Ubiquitination level of PFKFB4 protein was reduced in 293T cells in the presence of si-FBXL7, as determined by IP assay. J Ubiquitination level of PFKFB4 protein was elevated in 293T cells in the presence of oe-FBXL7, as determined by IP assay. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: Cells under hypoxic conditions were treated with si-EZH2 + si-FBXL7 or combined with 2-DG (16 mM, 154-17-6, MCE).

Techniques: Expressing, Co-Immunoprecipitation Assay, Pull Down Assay, Quantitative RT-PCR, Western Blot

A Western blot analysis of FBXL7 and PFKFB4 proteins in A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. B Glucose uptake measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. C Pyruvate level measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. D Lactate production measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. E ATP level measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. F ROS content measurement in A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. G ROS content measurement in mitochondrion in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. H Glycolysis of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG as detected by ECAR assay ( # p < 0.05 vs. the oe-FBXL7 group; @ p < 0.05 vs. the oe-FBXL7 + oe-PFKFB4 group). I Viability of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by CCK-8 assay. J Migration and invasion of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by Transwell assay. K Apoptosis of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A Western blot analysis of FBXL7 and PFKFB4 proteins in A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. B Glucose uptake measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. C Pyruvate level measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. D Lactate production measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. E ATP level measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. F ROS content measurement in A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. G ROS content measurement in mitochondrion in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. H Glycolysis of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG as detected by ECAR assay ( # p < 0.05 vs. the oe-FBXL7 group; @ p < 0.05 vs. the oe-FBXL7 + oe-PFKFB4 group). I Viability of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by CCK-8 assay. J Migration and invasion of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by Transwell assay. K Apoptosis of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: Cells under hypoxic conditions were treated with si-EZH2 + si-FBXL7 or combined with 2-DG (16 mM, 154-17-6, MCE).

Techniques: Western Blot, ECAR Assay, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry

A Transcriptional regulatory factors that bind to the FBXL7 promoter predicted by UCSC database, where EZH2 was found to be significantly enriched in the FBXL7 promoter region. B Expression of EZH2 mRNA was upregulated in NSCLC tissue samples, as analyzed by GEPIA database. Red box: tumor samples; gray box: normal samples; LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma. C Negative correlation between FBXL7 expression and EZH2 expression in NSCLC samples, as analyzed by GEPIA database. D FBXL7 mRNA expression was elevated in A549 and H1650 cells in the presence of si-EZH2, as determined by RT-qPCR. E Western blot analysis showing elevated FBXL7 protein expression in A549 and H1650 cells in the presence of si-EZH2. F Verification for the binding of EZH2 to the FBXL7 promoter region in A549 and H1650 cells, as determined by dual-luciferase reporter assay. G Enrichment of EZH2 in the promoter region of FBXL7 in A549 and H1650 cells, as observed by ChIP assay. ** p < 0.01, *** p < 0.001. ns p > 0.05. The cell ex p eriment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A Transcriptional regulatory factors that bind to the FBXL7 promoter predicted by UCSC database, where EZH2 was found to be significantly enriched in the FBXL7 promoter region. B Expression of EZH2 mRNA was upregulated in NSCLC tissue samples, as analyzed by GEPIA database. Red box: tumor samples; gray box: normal samples; LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma. C Negative correlation between FBXL7 expression and EZH2 expression in NSCLC samples, as analyzed by GEPIA database. D FBXL7 mRNA expression was elevated in A549 and H1650 cells in the presence of si-EZH2, as determined by RT-qPCR. E Western blot analysis showing elevated FBXL7 protein expression in A549 and H1650 cells in the presence of si-EZH2. F Verification for the binding of EZH2 to the FBXL7 promoter region in A549 and H1650 cells, as determined by dual-luciferase reporter assay. G Enrichment of EZH2 in the promoter region of FBXL7 in A549 and H1650 cells, as observed by ChIP assay. ** p < 0.01, *** p < 0.001. ns p > 0.05. The cell ex p eriment was repeated three times independently.

Article Snippet: Cells under hypoxic conditions were treated with si-EZH2 + si-FBXL7 or combined with 2-DG (16 mM, 154-17-6, MCE).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Binding Assay, Luciferase, Reporter Assay

A Western blot analysis showing elevated expression of EZH2 and HIF-1α proteins in hypoxic A549 cells. B Verification of the binding of HIF-1α to the EZH2 promoter region, as evaluated by dual-luciferase reporter assay. C Enrichment of HIF-1α in the promoter region of EZH2, as observed by ChIP assay. D mRNA expression of EZH2, FBXL7, and PFKFB4 in normoxic or hypoxic A549 cells in the presence of si-EZH2. E Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in normoxic or hypoxic A549 cells in the presence of si-EZH2. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A Western blot analysis showing elevated expression of EZH2 and HIF-1α proteins in hypoxic A549 cells. B Verification of the binding of HIF-1α to the EZH2 promoter region, as evaluated by dual-luciferase reporter assay. C Enrichment of HIF-1α in the promoter region of EZH2, as observed by ChIP assay. D mRNA expression of EZH2, FBXL7, and PFKFB4 in normoxic or hypoxic A549 cells in the presence of si-EZH2. E Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in normoxic or hypoxic A549 cells in the presence of si-EZH2. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: Cells under hypoxic conditions were treated with si-EZH2 + si-FBXL7 or combined with 2-DG (16 mM, 154-17-6, MCE).

Techniques: Western Blot, Expressing, Binding Assay, Luciferase, Reporter Assay

A The mRNA expression of EZH2, FBXL7 and PFKFB4 in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. B Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. C Glucose uptake measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. D Pyruvate level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. E Lactate production measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. F ATP level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. G ROS content measurement in A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. H ROS content measurement in mitochondrion in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. I Glycolysis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as detected by ECAR assay ( # p < 0.05 vs. the Hypoxia + si-EZH2 group; @ p < 0.05 vs. the Hypoxia + si-NC + si-FBXL7 group). J Viability of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by CCK-8 assay. K Migration and invasion of A549 cells, as analyzed by Transwell assay. L Apoptosis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A The mRNA expression of EZH2, FBXL7 and PFKFB4 in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. B Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. C Glucose uptake measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. D Pyruvate level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. E Lactate production measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. F ATP level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. G ROS content measurement in A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. H ROS content measurement in mitochondrion in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. I Glycolysis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as detected by ECAR assay ( # p < 0.05 vs. the Hypoxia + si-EZH2 group; @ p < 0.05 vs. the Hypoxia + si-NC + si-FBXL7 group). J Viability of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by CCK-8 assay. K Migration and invasion of A549 cells, as analyzed by Transwell assay. L Apoptosis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: Cells under hypoxic conditions were treated with si-EZH2 + si-FBXL7 or combined with 2-DG (16 mM, 154-17-6, MCE).

Techniques: Expressing, Western Blot, ECAR Assay, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry

A Quantitative analysis for tumor volume of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. B Quantitative analysis for tumor weight of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. C HE staining analysis of the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. D Immunohistochemical staining analysis of Ki-67, EZH2, PFKFB4, and FBXL7 proteins in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. E TUNEL-positive cells in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. *** p < 0.001, **** p < 0.0001. ns p > 0.05. n = 6 for mice in each group.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A Quantitative analysis for tumor volume of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. B Quantitative analysis for tumor weight of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. C HE staining analysis of the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. D Immunohistochemical staining analysis of Ki-67, EZH2, PFKFB4, and FBXL7 proteins in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. E TUNEL-positive cells in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. *** p < 0.001, **** p < 0.0001. ns p > 0.05. n = 6 for mice in each group.

Article Snippet: Cells under hypoxic conditions were treated with si-EZH2 + si-FBXL7 or combined with 2-DG (16 mM, 154-17-6, MCE).

Techniques: Transduction, Staining, Immunohistochemical staining, TUNEL Assay

Hypoxia induces the expression of HIF-1α in NSCLC cells. HIF-1α elevates expression of EZH2 which in turn reduces expression of FBXL7, and inhibits the ubiquitination of PFKFB4 by FBXL7, resulting in upregulation of PFKFB4 protein expression. By this mechanism, NSCLC cell glycolysis and malignant phenotypes are accelerated while cell apoptosis was decelerated.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: Hypoxia induces the expression of HIF-1α in NSCLC cells. HIF-1α elevates expression of EZH2 which in turn reduces expression of FBXL7, and inhibits the ubiquitination of PFKFB4 by FBXL7, resulting in upregulation of PFKFB4 protein expression. By this mechanism, NSCLC cell glycolysis and malignant phenotypes are accelerated while cell apoptosis was decelerated.

Article Snippet: Cells under hypoxic conditions were treated with si-EZH2 + si-FBXL7 or combined with 2-DG (16 mM, 154-17-6, MCE).

Techniques: Expressing

Fig. 6 Hypoxia increases the expression of EZH2, which reduces the expression of FBXL7 and promotes PFKFB4 protein stability. A Western blot analysis showing elevated expression of EZH2 and HIF-1α proteins in hypoxic A549 cells. B Verification of the binding of HIF-1α to the EZH2 promoter region, as evaluated by dual-luciferase reporter assay. C Enrichment of HIF-1α in the promoter region of EZH2, as observed by ChIP assay. D mRNA expression of EZH2, FBXL7, and PFKFB4 in normoxic or hypoxic A549 cells in the presence of si-EZH2. E Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in normoxic or hypoxic A549 cells in the presence of si- EZH2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell death & disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis.

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: Fig. 6 Hypoxia increases the expression of EZH2, which reduces the expression of FBXL7 and promotes PFKFB4 protein stability. A Western blot analysis showing elevated expression of EZH2 and HIF-1α proteins in hypoxic A549 cells. B Verification of the binding of HIF-1α to the EZH2 promoter region, as evaluated by dual-luciferase reporter assay. C Enrichment of HIF-1α in the promoter region of EZH2, as observed by ChIP assay. D mRNA expression of EZH2, FBXL7, and PFKFB4 in normoxic or hypoxic A549 cells in the presence of si-EZH2. E Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in normoxic or hypoxic A549 cells in the presence of si- EZH2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: The immunohistochemical staining included primary antibodies against FBXL7 (sc-374319, 1:100, Santa Cruz), Ki-67 (ab15580, 1:100, Abcam), EZH2 (5246, 1:100, CST), and PFKFB4 (ab137785, 1:100, Abcam).

Techniques: Expressing, Western Blot, Binding Assay, Luciferase, Reporter Assay

Fig. 7 Hypoxia accelerates glucose metabolism and the malignant phenotype of NSCLC cells via the EZH2/FBXL7/PFKFB4 axis. A The mRNA expression of EZH2, FBXL7 and PFKFB4 in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si- FBXL7 + 2-DG. B Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. C Glucose uptake measurement in response to si-EZH2, si-EZH2 + si- FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. D Pyruvate level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. E Lactate production measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. F ATP level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. G ROS content measurement in A549 cells in response to si-EZH2, si- EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. H ROS content measurement in mitochondrion in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. I Glycolysis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as detected by ECAR assay (#p < 0.05 vs. the Hypoxia + si-EZH2 group; @p < 0.05 vs. the Hypoxia + si-NC + si-FBXL7 group). J Viability of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by CCK-8 assay. K Migration and invasion of A549 cells, as analyzed by Transwell assay. L Apoptosis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell death & disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis.

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: Fig. 7 Hypoxia accelerates glucose metabolism and the malignant phenotype of NSCLC cells via the EZH2/FBXL7/PFKFB4 axis. A The mRNA expression of EZH2, FBXL7 and PFKFB4 in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si- FBXL7 + 2-DG. B Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. C Glucose uptake measurement in response to si-EZH2, si-EZH2 + si- FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. D Pyruvate level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. E Lactate production measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. F ATP level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. G ROS content measurement in A549 cells in response to si-EZH2, si- EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. H ROS content measurement in mitochondrion in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. I Glycolysis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as detected by ECAR assay (#p < 0.05 vs. the Hypoxia + si-EZH2 group; @p < 0.05 vs. the Hypoxia + si-NC + si-FBXL7 group). J Viability of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by CCK-8 assay. K Migration and invasion of A549 cells, as analyzed by Transwell assay. L Apoptosis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: The immunohistochemical staining included primary antibodies against FBXL7 (sc-374319, 1:100, Santa Cruz), Ki-67 (ab15580, 1:100, Abcam), EZH2 (5246, 1:100, CST), and PFKFB4 (ab137785, 1:100, Abcam).

Techniques: Expressing, Western Blot, ECAR Assay, CCK-8 Assay, Migration, Transwell Assay, Cytometry

Fig. 8 Knockdown of EZH2 retards tumor growth in vivo via the FBXL7/PFKFB4 axis. A Quantitative analysis for tumor volume of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. B Quantitative analysis for tumor weight of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. C HE staining analysis of the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. D Immunohistochemical staining analysis of Ki-67, EZH2, PFKFB4, and FBXL7 proteins in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. E TUNEL-positive cells in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. ***p < 0.001, ****p < 0.0001. ns p > 0.05. n = 6 for mice in each group.

Journal: Cell death & disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis.

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: Fig. 8 Knockdown of EZH2 retards tumor growth in vivo via the FBXL7/PFKFB4 axis. A Quantitative analysis for tumor volume of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. B Quantitative analysis for tumor weight of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. C HE staining analysis of the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. D Immunohistochemical staining analysis of Ki-67, EZH2, PFKFB4, and FBXL7 proteins in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. E TUNEL-positive cells in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. ***p < 0.001, ****p < 0.0001. ns p > 0.05. n = 6 for mice in each group.

Article Snippet: The immunohistochemical staining included primary antibodies against FBXL7 (sc-374319, 1:100, Santa Cruz), Ki-67 (ab15580, 1:100, Abcam), EZH2 (5246, 1:100, CST), and PFKFB4 (ab137785, 1:100, Abcam).

Techniques: Knockdown, In Vivo, Transduction, Staining, Immunohistochemical staining, TUNEL Assay

Fig. 9 Schematic mechanism of hypoxia-mediated EZH2/FBXL7/PFKFB4 axis in NSCLC. Hypoxia induces the expression of HIF-1α in NSCLC cells. HIF-1α elevates expression of EZH2 which in turn reduces expression of FBXL7, and inhibits the ubiquitination of PFKFB4 by FBXL7, resulting in upregulation of PFKFB4 protein expression. By this mechanism, NSCLC cell glycolysis and malignant phenotypes are accelerated while cell apoptosis was decelerated.

Journal: Cell death & disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis.

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: Fig. 9 Schematic mechanism of hypoxia-mediated EZH2/FBXL7/PFKFB4 axis in NSCLC. Hypoxia induces the expression of HIF-1α in NSCLC cells. HIF-1α elevates expression of EZH2 which in turn reduces expression of FBXL7, and inhibits the ubiquitination of PFKFB4 by FBXL7, resulting in upregulation of PFKFB4 protein expression. By this mechanism, NSCLC cell glycolysis and malignant phenotypes are accelerated while cell apoptosis was decelerated.

Article Snippet: The immunohistochemical staining included primary antibodies against FBXL7 (sc-374319, 1:100, Santa Cruz), Ki-67 (ab15580, 1:100, Abcam), EZH2 (5246, 1:100, CST), and PFKFB4 (ab137785, 1:100, Abcam).

Techniques: Expressing, Ubiquitin Proteomics

A PFKFB4 expression was elevated in NSCLC tissue samples, as analyzed by UALCAN database. LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma. B Kaplan–Meier analysis showing the negative correlation between PFKFB4 and the overall survival rate of NSCLC patients. Red: high expression of PFKFB4; black: low expression of PFKFB4. C Co-IP analysis of the interaction of endogenous FBXL7 with PFKFB4 in 293T cells. D Co-IP analysis of the interaction of exogenous FBXL7 with PFKFB4 in 293T cells. E GST-pull down assay analysis of the direct interaction between FBXL7 and PFKFB4 in 293T cells. F PFKFB4 mRNA expression in A549 cells wasn’t changed in response to oe-FBXL7, as determined by RT-qPCR. G Western blot analysis showing inhibited PFKFB4 protein expression in A549 cells in response to oe-FBXL7. H Western blot analysis showing enhanced degradation of PFKFB4 protein in 293T cells in response to oe-FBXL7 and CHX. I Ubiquitination level of PFKFB4 protein was reduced in 293T cells in the presence of si-FBXL7, as determined by IP assay. J Ubiquitination level of PFKFB4 protein was elevated in 293T cells in the presence of oe-FBXL7, as determined by IP assay. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A PFKFB4 expression was elevated in NSCLC tissue samples, as analyzed by UALCAN database. LUSC: lung squamous cell carcinoma; LUAD: lung adenocarcinoma. B Kaplan–Meier analysis showing the negative correlation between PFKFB4 and the overall survival rate of NSCLC patients. Red: high expression of PFKFB4; black: low expression of PFKFB4. C Co-IP analysis of the interaction of endogenous FBXL7 with PFKFB4 in 293T cells. D Co-IP analysis of the interaction of exogenous FBXL7 with PFKFB4 in 293T cells. E GST-pull down assay analysis of the direct interaction between FBXL7 and PFKFB4 in 293T cells. F PFKFB4 mRNA expression in A549 cells wasn’t changed in response to oe-FBXL7, as determined by RT-qPCR. G Western blot analysis showing inhibited PFKFB4 protein expression in A549 cells in response to oe-FBXL7. H Western blot analysis showing enhanced degradation of PFKFB4 protein in 293T cells in response to oe-FBXL7 and CHX. I Ubiquitination level of PFKFB4 protein was reduced in 293T cells in the presence of si-FBXL7, as determined by IP assay. J Ubiquitination level of PFKFB4 protein was elevated in 293T cells in the presence of oe-FBXL7, as determined by IP assay. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: The immunohistochemical staining included primary antibodies against FBXL7 (sc-374319, 1:100, Santa Cruz), Ki-67 (ab15580, 1:100, Abcam), EZH2 (5246, 1:100, CST), and PFKFB4 (ab137785, 1:100, Abcam).

Techniques: Expressing, Co-Immunoprecipitation Assay, Pull Down Assay, Quantitative RT-PCR, Western Blot

A Western blot analysis of FBXL7 and PFKFB4 proteins in A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. B Glucose uptake measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. C Pyruvate level measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. D Lactate production measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. E ATP level measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. F ROS content measurement in A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. G ROS content measurement in mitochondrion in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. H Glycolysis of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG as detected by ECAR assay ( # p < 0.05 vs. the oe-FBXL7 group; @ p < 0.05 vs. the oe-FBXL7 + oe-PFKFB4 group). I Viability of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by CCK-8 assay. J Migration and invasion of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by Transwell assay. K Apoptosis of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A Western blot analysis of FBXL7 and PFKFB4 proteins in A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. B Glucose uptake measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. C Pyruvate level measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. D Lactate production measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. E ATP level measurement in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. F ROS content measurement in A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. G ROS content measurement in mitochondrion in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG. H Glycolysis of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG as detected by ECAR assay ( # p < 0.05 vs. the oe-FBXL7 group; @ p < 0.05 vs. the oe-FBXL7 + oe-PFKFB4 group). I Viability of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by CCK-8 assay. J Migration and invasion of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by Transwell assay. K Apoptosis of A549 cells in response to oe-FBXL7, oe-FBXL7 + oe-PFKFB4, or oe-FBXL7 + oe-PFKFB4 + 2-DG, as analyzed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: The immunohistochemical staining included primary antibodies against FBXL7 (sc-374319, 1:100, Santa Cruz), Ki-67 (ab15580, 1:100, Abcam), EZH2 (5246, 1:100, CST), and PFKFB4 (ab137785, 1:100, Abcam).

Techniques: Western Blot, ECAR Assay, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry

A Western blot analysis showing elevated expression of EZH2 and HIF-1α proteins in hypoxic A549 cells. B Verification of the binding of HIF-1α to the EZH2 promoter region, as evaluated by dual-luciferase reporter assay. C Enrichment of HIF-1α in the promoter region of EZH2, as observed by ChIP assay. D mRNA expression of EZH2, FBXL7, and PFKFB4 in normoxic or hypoxic A549 cells in the presence of si-EZH2. E Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in normoxic or hypoxic A549 cells in the presence of si-EZH2. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A Western blot analysis showing elevated expression of EZH2 and HIF-1α proteins in hypoxic A549 cells. B Verification of the binding of HIF-1α to the EZH2 promoter region, as evaluated by dual-luciferase reporter assay. C Enrichment of HIF-1α in the promoter region of EZH2, as observed by ChIP assay. D mRNA expression of EZH2, FBXL7, and PFKFB4 in normoxic or hypoxic A549 cells in the presence of si-EZH2. E Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in normoxic or hypoxic A549 cells in the presence of si-EZH2. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: The immunohistochemical staining included primary antibodies against FBXL7 (sc-374319, 1:100, Santa Cruz), Ki-67 (ab15580, 1:100, Abcam), EZH2 (5246, 1:100, CST), and PFKFB4 (ab137785, 1:100, Abcam).

Techniques: Western Blot, Expressing, Binding Assay, Luciferase, Reporter Assay

A The mRNA expression of EZH2, FBXL7 and PFKFB4 in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. B Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. C Glucose uptake measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. D Pyruvate level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. E Lactate production measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. F ATP level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. G ROS content measurement in A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. H ROS content measurement in mitochondrion in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. I Glycolysis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as detected by ECAR assay ( # p < 0.05 vs. the Hypoxia + si-EZH2 group; @ p < 0.05 vs. the Hypoxia + si-NC + si-FBXL7 group). J Viability of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by CCK-8 assay. K Migration and invasion of A549 cells, as analyzed by Transwell assay. L Apoptosis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A The mRNA expression of EZH2, FBXL7 and PFKFB4 in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. B Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in hypoxia-exposed A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. C Glucose uptake measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. D Pyruvate level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. E Lactate production measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. F ATP level measurement in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. G ROS content measurement in A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG. H ROS content measurement in mitochondrion in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG. I Glycolysis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as detected by ECAR assay ( # p < 0.05 vs. the Hypoxia + si-EZH2 group; @ p < 0.05 vs. the Hypoxia + si-NC + si-FBXL7 group). J Viability of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7 or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by CCK-8 assay. K Migration and invasion of A549 cells, as analyzed by Transwell assay. L Apoptosis of A549 cells in response to si-EZH2, si-EZH2 + si-FBXL7, or si-EZH2 + si-FBXL7 + 2-DG, as analyzed by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: The immunohistochemical staining included primary antibodies against FBXL7 (sc-374319, 1:100, Santa Cruz), Ki-67 (ab15580, 1:100, Abcam), EZH2 (5246, 1:100, CST), and PFKFB4 (ab137785, 1:100, Abcam).

Techniques: Expressing, Western Blot, ECAR Assay, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry

A Quantitative analysis for tumor volume of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. B Quantitative analysis for tumor weight of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. C HE staining analysis of the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. D Immunohistochemical staining analysis of Ki-67, EZH2, PFKFB4, and FBXL7 proteins in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. E TUNEL-positive cells in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. *** p < 0.001, **** p < 0.0001. ns p > 0.05. n = 6 for mice in each group.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A Quantitative analysis for tumor volume of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. B Quantitative analysis for tumor weight of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. C HE staining analysis of the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. D Immunohistochemical staining analysis of Ki-67, EZH2, PFKFB4, and FBXL7 proteins in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. E TUNEL-positive cells in the tumor tissues of nude mice subcutaneously inoculated with cells transduced with sh-EZH2 alone or combined with oe-PFKFB4. *** p < 0.001, **** p < 0.0001. ns p > 0.05. n = 6 for mice in each group.

Article Snippet: The immunohistochemical staining included primary antibodies against FBXL7 (sc-374319, 1:100, Santa Cruz), Ki-67 (ab15580, 1:100, Abcam), EZH2 (5246, 1:100, CST), and PFKFB4 (ab137785, 1:100, Abcam).

Techniques: Transduction, Staining, Immunohistochemical staining, TUNEL Assay

Hypoxia induces the expression of HIF-1α in NSCLC cells. HIF-1α elevates expression of EZH2 which in turn reduces expression of FBXL7, and inhibits the ubiquitination of PFKFB4 by FBXL7, resulting in upregulation of PFKFB4 protein expression. By this mechanism, NSCLC cell glycolysis and malignant phenotypes are accelerated while cell apoptosis was decelerated.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: Hypoxia induces the expression of HIF-1α in NSCLC cells. HIF-1α elevates expression of EZH2 which in turn reduces expression of FBXL7, and inhibits the ubiquitination of PFKFB4 by FBXL7, resulting in upregulation of PFKFB4 protein expression. By this mechanism, NSCLC cell glycolysis and malignant phenotypes are accelerated while cell apoptosis was decelerated.

Article Snippet: The immunohistochemical staining included primary antibodies against FBXL7 (sc-374319, 1:100, Santa Cruz), Ki-67 (ab15580, 1:100, Abcam), EZH2 (5246, 1:100, CST), and PFKFB4 (ab137785, 1:100, Abcam).

Techniques: Expressing

A Western blot analysis showing elevated expression of EZH2 and HIF-1α proteins in hypoxic A549 cells. B Verification of the binding of HIF-1α to the EZH2 promoter region, as evaluated by dual-luciferase reporter assay. C Enrichment of HIF-1α in the promoter region of EZH2, as observed by ChIP assay. D mRNA expression of EZH2, FBXL7, and PFKFB4 in normoxic or hypoxic A549 cells in the presence of si-EZH2. E Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in normoxic or hypoxic A549 cells in the presence of si-EZH2. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: A Western blot analysis showing elevated expression of EZH2 and HIF-1α proteins in hypoxic A549 cells. B Verification of the binding of HIF-1α to the EZH2 promoter region, as evaluated by dual-luciferase reporter assay. C Enrichment of HIF-1α in the promoter region of EZH2, as observed by ChIP assay. D mRNA expression of EZH2, FBXL7, and PFKFB4 in normoxic or hypoxic A549 cells in the presence of si-EZH2. E Western blot analysis showing the expression of EZH2, FBXL7, and PFKFB4 proteins in normoxic or hypoxic A549 cells in the presence of si-EZH2. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns p > 0.05. The cell experiment was repeated three times independently.

Article Snippet: After being blocked using 5% BSA, membranes underwent overnight incubation at 4 °C with the diluted primary antibodies against EZH2 (5246, 1:1000, Cell Signaling Technologies [CST], Beverly, MA), FBXL7 (sc-374319, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), HA (ab9110, 1:1000, Abcam, Cambridge, UK), PFKFB4 (ab137785, 1:1000, Abcam), HIF-1α (36169, 1:1000, CST), Myc (ab32, 1:1000, Abcam) and Actin (ab8226, 1:2000, Abcam).

Techniques: Western Blot, Expressing, Binding Assay, Luciferase, Reporter Assay

Hypoxia induces the expression of HIF-1α in NSCLC cells. HIF-1α elevates expression of EZH2 which in turn reduces expression of FBXL7, and inhibits the ubiquitination of PFKFB4 by FBXL7, resulting in upregulation of PFKFB4 protein expression. By this mechanism, NSCLC cell glycolysis and malignant phenotypes are accelerated while cell apoptosis was decelerated.

Journal: Cell Death & Disease

Article Title: Hypoxia-mediated promotion of glucose metabolism in non-small cell lung cancer correlates with activation of the EZH2/FBXL7/PFKFB4 axis

doi: 10.1038/s41419-023-05795-z

Figure Lengend Snippet: Hypoxia induces the expression of HIF-1α in NSCLC cells. HIF-1α elevates expression of EZH2 which in turn reduces expression of FBXL7, and inhibits the ubiquitination of PFKFB4 by FBXL7, resulting in upregulation of PFKFB4 protein expression. By this mechanism, NSCLC cell glycolysis and malignant phenotypes are accelerated while cell apoptosis was decelerated.

Article Snippet: After being blocked using 5% BSA, membranes underwent overnight incubation at 4 °C with the diluted primary antibodies against EZH2 (5246, 1:1000, Cell Signaling Technologies [CST], Beverly, MA), FBXL7 (sc-374319, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), HA (ab9110, 1:1000, Abcam, Cambridge, UK), PFKFB4 (ab137785, 1:1000, Abcam), HIF-1α (36169, 1:1000, CST), Myc (ab32, 1:1000, Abcam) and Actin (ab8226, 1:2000, Abcam).

Techniques: Expressing, Ubiquitin Proteomics