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rabbit anti-human/mouse hk2 (Cell Signaling Technology Inc)

Name: rabbit anti-human/mouse hk2
Brand: Cell Signaling Technology Inc
Rating: 90 (1 reviews)

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Cell Signaling Technology Inc rabbit anti-human/mouse hk2
Rabbit Anti Human/Mouse Hk2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit anti-human/mouse hk2/product/Cell Signaling Technology Inc
Average 90 stars, based on 1 article reviews

rabbit anti-human/mouse hk2 - by Bioz Stars, 2026-02

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HK2 translocates to mitochondria after CoCl 2 and PDGF stimulation. (A) Confocal microscopy of RA FLS (n = 3) after stimulation with 150 µM cobalt chloride (CoCl 2 ) for 30 and 60 min with quantification. HK2 protein is stained with green fluorescence, ATP5a is stained with red fluorescence, and DAPI is stained with blue fluorescence as control. Overlapping yellow color indicates HK2 colocalization to mitochondria. n = 3 independent experiments. The comparison between groups was performed using unpaired two-tailed Student’s t -test. Statistical significance was considered when p-value ≤ 0.05. (B) Confocal microscopy of RA FLS (n = 3) after stimulation of PDGF (20 ng/ml) after 30 and 60 min with quantification. HK2 protein is stained with green fluorescence and ATP5a is stained with red fluorescence. Overlapping yellow color indicates HK2 colocalization to mitochondria. n = 2 independent experiments. The comparison between groups was performed using unpaired two-tailed student t -test. Statistical significance was considered when p-value ≤ 0.05. *P ≤ 0.05.

Journal: Frontiers in Immunology

Article Title: Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes

doi: 10.3389/fimmu.2023.1103231

Figure Lengend Snippet: HK2 translocates to mitochondria after CoCl 2 and PDGF stimulation. (A) Confocal microscopy of RA FLS (n = 3) after stimulation with 150 µM cobalt chloride (CoCl 2 ) for 30 and 60 min with quantification. HK2 protein is stained with green fluorescence, ATP5a is stained with red fluorescence, and DAPI is stained with blue fluorescence as control. Overlapping yellow color indicates HK2 colocalization to mitochondria. n = 3 independent experiments. The comparison between groups was performed using unpaired two-tailed Student’s t -test. Statistical significance was considered when p-value ≤ 0.05. (B) Confocal microscopy of RA FLS (n = 3) after stimulation of PDGF (20 ng/ml) after 30 and 60 min with quantification. HK2 protein is stained with green fluorescence and ATP5a is stained with red fluorescence. Overlapping yellow color indicates HK2 colocalization to mitochondria. n = 2 independent experiments. The comparison between groups was performed using unpaired two-tailed student t -test. Statistical significance was considered when p-value ≤ 0.05. *P ≤ 0.05.

Article Snippet: After blocking for 1 h with 5% milk, blots were incubated overnight at 4 ° C with the following antibodies: rabbit anti-human/mouse HK2 at 1:500 (#2867, C64G5, Cell Signaling Technology, Danvers, MA), mouse anti-human HK2 at 1:500 (sc-374091, B8, Santa Cruz Co, Santa Clara, CA), mouse anti-human tubulin at 1:1,000 (Santa Cruz Co., Santa Clara, CA), rabbit anti-TOM20 at 1:500 (Cell Signaling Technology, Danvers, MA), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and tubulin (Santa Cruz Co., Santa Clara, CA).

Techniques: Confocal Microscopy, Staining, Fluorescence, Control, Comparison, Two Tailed Test

Effect of mitochondrial mutant HK2 on RA FLS invasion and migration. (A, B) RA FLSs were plated in matrigel spheroids as described in methods after adenovirus infection with Ad-GFP control, Ad-HK2FL, and Ad- HK2ΔN constructs for 2 days. (A) Analysis of area of invasion after infection with adenovirus. The comparison between groups was performed using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical analysis of P-value ≤ 0.05 was considered significant. (B) Representative images of invasion with the adenovirus constructs with and without PDGF. (C, D) RA FLSs were plated in six-well plates and scratched with the end of a sterile pipette tip after infection with adenovirus constructs. Cells were left to migrate 24 h with and without PDGF (10 ng/ml) stimulation. (C) Quantification of scratch length. The comparison between groups was performed using Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Statistical significance was considered when p-value ≤ 0.05. (D) Representative images of migration with different adenovirus constructs with and without PDGF stimulation. ****P ≤ 0.001; **P ≤ 0.01.

Journal: Frontiers in Immunology

Article Title: Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes

doi: 10.3389/fimmu.2023.1103231

Figure Lengend Snippet: Effect of mitochondrial mutant HK2 on RA FLS invasion and migration. (A, B) RA FLSs were plated in matrigel spheroids as described in methods after adenovirus infection with Ad-GFP control, Ad-HK2FL, and Ad- HK2ΔN constructs for 2 days. (A) Analysis of area of invasion after infection with adenovirus. The comparison between groups was performed using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical analysis of P-value ≤ 0.05 was considered significant. (B) Representative images of invasion with the adenovirus constructs with and without PDGF. (C, D) RA FLSs were plated in six-well plates and scratched with the end of a sterile pipette tip after infection with adenovirus constructs. Cells were left to migrate 24 h with and without PDGF (10 ng/ml) stimulation. (C) Quantification of scratch length. The comparison between groups was performed using Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Statistical significance was considered when p-value ≤ 0.05. (D) Representative images of migration with different adenovirus constructs with and without PDGF stimulation. ****P ≤ 0.001; **P ≤ 0.01. "ns" denotes "not significant".

Techniques: Mutagenesis, Migration, Infection, Control, Construct, Comparison, Sterility, Transferring

Treatment with methyl jasmonate (MJ), which dissociates HK2 from mitochondria, impaired FLS invasion and migration. (A, B) RA FLSs (n = 4) were plated in matrigel spheroids and given vehicle (ethanol) or MJ 1 h before PDGF stimulation. (A) Quantification of area of invasion after MJ treatment. Comparison between groups was performed using Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis of P-value ≤ 0.05 was considered significant. (B) Representative images of invasion with MJ. (C, D) RA FLSs were plated in six-well plates and scratched with the end of a sterile pipette tip. Cells were given vehicle (ethanol) or MJ 1 h before PDGF stimulation and allowed to migrate for 24 h, (C) Quantification of scratch length. The comparison between groups was performed using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical analysis of P-value ≤ 0.05 was considered significant. (D) Representative images of MJ migration. (E, F) RA FLSs were plated in 12-well plates and treated with vehicle (ethanol) or MJ 1 h before addition of 0 µM H 2 O 2 , 75 µM H 2 O 2 , and 200 µM H 2 O 2 to assess cell death and were fixed and stained after 4 h, (E) Analysis of mean gray value of cells using Fiji software. Comparison between groups was performed using Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis of P-value ≤ 0.05 was considered significant. (F) Representative images of control and 1.5 mM MJ. *P ≤ 0.05.

Journal: Frontiers in Immunology

Article Title: Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes

doi: 10.3389/fimmu.2023.1103231

Figure Lengend Snippet: Treatment with methyl jasmonate (MJ), which dissociates HK2 from mitochondria, impaired FLS invasion and migration. (A, B) RA FLSs (n = 4) were plated in matrigel spheroids and given vehicle (ethanol) or MJ 1 h before PDGF stimulation. (A) Quantification of area of invasion after MJ treatment. Comparison between groups was performed using Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis of P-value ≤ 0.05 was considered significant. (B) Representative images of invasion with MJ. (C, D) RA FLSs were plated in six-well plates and scratched with the end of a sterile pipette tip. Cells were given vehicle (ethanol) or MJ 1 h before PDGF stimulation and allowed to migrate for 24 h, (C) Quantification of scratch length. The comparison between groups was performed using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical analysis of P-value ≤ 0.05 was considered significant. (D) Representative images of MJ migration. (E, F) RA FLSs were plated in 12-well plates and treated with vehicle (ethanol) or MJ 1 h before addition of 0 µM H 2 O 2 , 75 µM H 2 O 2 , and 200 µM H 2 O 2 to assess cell death and were fixed and stained after 4 h, (E) Analysis of mean gray value of cells using Fiji software. Comparison between groups was performed using Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis of P-value ≤ 0.05 was considered significant. (F) Representative images of control and 1.5 mM MJ. *P ≤ 0.05. "ns" denotes "not significant".

Techniques: Migration, Comparison, Sterility, Transferring, Staining, Software, Control

Effect of common RA drugs on mitochondrial translocation of HK2. Confocal microscopy of RA FLS (n = 3) after 60 min of stimulation of 150 µM CoCl 2 with and without either 1 µM tofacitinib for 60 min or 1 µM MTX overnight with quantification. HK2 protein is stained with green fluorescence, and ATP5a is stained with red fluorescence. Overlapping yellow color indicates HK2 colocalization to mitochondria. n = 3 independent experiments. The comparison between groups was performed using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical analysis of P-value ≤ 0.05 was considered significant. *P ≤ 0.05.

Journal: Frontiers in Immunology

Article Title: Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes

doi: 10.3389/fimmu.2023.1103231

Figure Lengend Snippet: Effect of common RA drugs on mitochondrial translocation of HK2. Confocal microscopy of RA FLS (n = 3) after 60 min of stimulation of 150 µM CoCl 2 with and without either 1 µM tofacitinib for 60 min or 1 µM MTX overnight with quantification. HK2 protein is stained with green fluorescence, and ATP5a is stained with red fluorescence. Overlapping yellow color indicates HK2 colocalization to mitochondria. n = 3 independent experiments. The comparison between groups was performed using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical analysis of P-value ≤ 0.05 was considered significant. *P ≤ 0.05.

Techniques: Translocation Assay, Confocal Microscopy, Staining, Fluorescence, Comparison

MJ decreased the expression of genes associated to HK2 identified by scRNA-seq. (A) UMAP of scRNAseq data of live CD45 − cells from digested hind limbs with main cell labels (n = 3 mice). Vasc, vascular cells; Peri. Vasc, perivascular; Contam., contamination; Osteo., osteoblasts; Chondr., chondrocytes. (B) Fibroblast subsets with cell labels based on reads of Hk2 gene. (C) Gene expression (GEX) of top 10 marker genes in Hk2-positive cells compared with Hk2 -egative cells with GO term. (D) Heatmap of differential expressed genes between disease model time point (rest, resolved, peak, and resolving) in Hk2 -positive cells. (E) Volcano plots of differentially expressed genes between the indicated conditional in Hk2 -positive cells. Each dot represents a gene. Genes in red: adjusted p-value < 0.05 and fold change > 0.5 and < −0.5. P-value calculated using FindMarkers() (Seurat) and the Wilcox method. (F) qPCR analysis of the indicated genes in RA FLS 6 h of MJ treatment. Results are average of three different RA FLS lines. Statistical analysis of P-value ≤ 0.05 was considered significant. *P ≤ 0.05 ; **P ≤ 0.01; ***P ≤ 0.001.

Journal: Frontiers in Immunology

Article Title: Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes

doi: 10.3389/fimmu.2023.1103231

Figure Lengend Snippet: MJ decreased the expression of genes associated to HK2 identified by scRNA-seq. (A) UMAP of scRNAseq data of live CD45 − cells from digested hind limbs with main cell labels (n = 3 mice). Vasc, vascular cells; Peri. Vasc, perivascular; Contam., contamination; Osteo., osteoblasts; Chondr., chondrocytes. (B) Fibroblast subsets with cell labels based on reads of Hk2 gene. (C) Gene expression (GEX) of top 10 marker genes in Hk2-positive cells compared with Hk2 -egative cells with GO term. (D) Heatmap of differential expressed genes between disease model time point (rest, resolved, peak, and resolving) in Hk2 -positive cells. (E) Volcano plots of differentially expressed genes between the indicated conditional in Hk2 -positive cells. Each dot represents a gene. Genes in red: adjusted p-value < 0.05 and fold change > 0.5 and < −0.5. P-value calculated using FindMarkers() (Seurat) and the Wilcox method. (F) qPCR analysis of the indicated genes in RA FLS 6 h of MJ treatment. Results are average of three different RA FLS lines. Statistical analysis of P-value ≤ 0.05 was considered significant. *P ≤ 0.05 ; **P ≤ 0.01; ***P ≤ 0.001.

Techniques: Expressing, Gene Expression, Marker

Adenovirus injection and Ad-15G treatment in AIA model. (A, B) B6 mice (n = 10) injected with AdenoHK2FL or Adeno HK2ΔN in either knee as described in methods. (A) Histological scores of mouse knees measuring synovial infiltration and infiltration of either HK2FL or HK2ΔN. The comparison between groups was performed using two-tailed Mann–Whitney non-parametric test for each score type. Statistical significance was considered when p-value ≤ 0.05. (B) Representative images of knee joints of mice. Asterisk (*) shows synovium (C, D) AIA mouse model (n = 10) with treatment of 15G peptide that dissociates HK2 from mitochondria as described in methods. (C) Histological score of mouse knees measuring synovial infiltration or infiltration of mice injected with Ad-GFP control or Ad-15G. The comparison between groups was performed using two-tailed Mann–Whitney non-parametric test for each score type. Statistical significance was considered when p-value ≤ 0.05. (D) Representative images of mouse joints.

Journal: Frontiers in Immunology

Article Title: Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes

doi: 10.3389/fimmu.2023.1103231

Figure Lengend Snippet: Adenovirus injection and Ad-15G treatment in AIA model. (A, B) B6 mice (n = 10) injected with AdenoHK2FL or Adeno HK2ΔN in either knee as described in methods. (A) Histological scores of mouse knees measuring synovial infiltration and infiltration of either HK2FL or HK2ΔN. The comparison between groups was performed using two-tailed Mann–Whitney non-parametric test for each score type. Statistical significance was considered when p-value ≤ 0.05. (B) Representative images of knee joints of mice. Asterisk (*) shows synovium (C, D) AIA mouse model (n = 10) with treatment of 15G peptide that dissociates HK2 from mitochondria as described in methods. (C) Histological score of mouse knees measuring synovial infiltration or infiltration of mice injected with Ad-GFP control or Ad-15G. The comparison between groups was performed using two-tailed Mann–Whitney non-parametric test for each score type. Statistical significance was considered when p-value ≤ 0.05. (D) Representative images of mouse joints.

Techniques: Injection, Comparison, Two Tailed Test, MANN-WHITNEY, Control

Journal: Cell host & microbe

Article Title: Legionella-infected macrophages engage the alveolar epithelium to metabolically reprogram myeloid cells and promote antibacterial inflammation

doi: 10.1016/j.chom.2020.07.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-Mouse/Human HK2 (clone C64G5) , Cell Signaling Technology , Cat#2867; RRID: AB_2232946.

Techniques: Virus, Recombinant, Reverse Transcription, Membrane, Isolation, Enzyme-linked Immunosorbent Assay, Plex Assay, Flow Cytometry, Software

Alterations in select sarcoplasmic enzymes with CON vs. VAR training. Data presented in arbitrary units (ADUs) include sarcoplasmic protein concentrations from biopsied muscle (panel A ), creatine kinase, M-type (CKM) protein levels from the sarcoplasmic protein pool (panel B ), hexokinase 2 (HK2) protein levels from the sarcoplasmic protein pool (panel C ), lactate dehydrogenase A (LDHA) protein levels from the sarcoplasmic protein pool (panel D ), phosphofructokinase (PFK) protein levels from the sarcoplasmic protein pool (panel E ), and glycogen phosphorylase (PYGM) protein levels from the sarcoplasmic protein pool (panel F ). Data are presented as means with standard deviation bars. Panel (G) contains representative Western blot images for each target. Note that two of Ponceau images in panel (G) (for HK2 and LDHA) are the same given that the same subject was used for the representative Western blot images of these targets.

Journal: Frontiers in Physiology

Article Title: Frequent Manipulation of Resistance Training Variables Promotes Myofibrillar Spacing Changes in Resistance-Trained Individuals

doi: 10.3389/fphys.2021.773995

Figure Lengend Snippet: Alterations in select sarcoplasmic enzymes with CON vs. VAR training. Data presented in arbitrary units (ADUs) include sarcoplasmic protein concentrations from biopsied muscle (panel A ), creatine kinase, M-type (CKM) protein levels from the sarcoplasmic protein pool (panel B ), hexokinase 2 (HK2) protein levels from the sarcoplasmic protein pool (panel C ), lactate dehydrogenase A (LDHA) protein levels from the sarcoplasmic protein pool (panel D ), phosphofructokinase (PFK) protein levels from the sarcoplasmic protein pool (panel E ), and glycogen phosphorylase (PYGM) protein levels from the sarcoplasmic protein pool (panel F ). Data are presented as means with standard deviation bars. Panel (G) contains representative Western blot images for each target. Note that two of Ponceau images in panel (G) (for HK2 and LDHA) are the same given that the same subject was used for the representative Western blot images of these targets.

Article Snippet: Membranes were then incubated overnight at 4°C with the following antibody cocktails (each at a 1:1,000 dilution) in TBST with 5% bovine serum albumin (BSA): (i) goat anti-human creatine kinase (CKM; Abcam; Cambridge, MA, United States; cat# ab174672), (ii) rabbit anti-human L-type amino acid transporter (LAT1, Cell Signaling Technology; Danvers, MA, United States; cat# 5347), (iii) rabbit anti-human glucose transporter 4 (GLUT4; Cell Signaling Technology, cat# 2213), (iv) rabbit anti-human lactate dehydrogenase (LDHA; Cell Signaling Technology, cat# 2012), (v) rabbit anti-human phosphofructokinase (PFKM; Abcam, cat#: ab154804), (vi) rabbit anti-human total oxidative phosphorylation (OXPHOS) cocktail (Abcam, cat# ab110411), (vii) rabbit anti-human complex IV (Cell Signaling Technology, cat# 4850), (viii) rabbit anti-human isocitrate dehydrogenase 2 (IDH2; Cell Signaling Technology, cat# 56439), (ix) rabbit anti-human acyl-CoA dehydrogenase very long chain (VLCAD; Abcam, cat# ab188872), (x) rabbit anti-human carnitine palmitoyltransferase I (CPTI; Abcam, cat# ab134135), and (xi) rabbit anti-human hexokinase II (HK2; Cell Signaling Technology, cat# 2867).

Techniques: Standard Deviation, Western Blot

The correlation of hsa-miR-9-5p and HK2 expression in patient samples and radio sensitive and resistant cells. (A) A schematic diagram for the study. (B) The mRNA expression levels of hsa-miR-9-5p and HK2 in NPC patients (n = 20). (C) The mRNA expression levels of hsa-miR-9-5p and HK2 in 5-8F and 58FR cells (n = 4) (** p < 0.01 vs. 5-8F).

Journal: Technology in Cancer Research & Treatment

Article Title: hsa-miR-9-5p Down-Regulates HK2 and Confers Radiosensitivity to Nasopharyngeal Carcinoma

doi: 10.1177/1533033821997822

Figure Lengend Snippet: The correlation of hsa-miR-9-5p and HK2 expression in patient samples and radio sensitive and resistant cells. (A) A schematic diagram for the study. (B) The mRNA expression levels of hsa-miR-9-5p and HK2 in NPC patients (n = 20). (C) The mRNA expression levels of hsa-miR-9-5p and HK2 in 5-8F and 58FR cells (n = 4) (** p < 0.01 vs. 5-8F).

Article Snippet: After being blocked with 4% bovine serum albumin (BSA) for 1 h, the membranes were incubated with rabbit anti-human HK2 antibody (AF7080, 1:250, Beyotime, Shanghai, China) overnight at 4 C. Then, the secondary antibody (1:1 000, Santa Cruz, California, USA) was incubated at 4°C for 1 h and the gel imaging system ECL (Invitrogen, USA) was used for luminescence development.

Techniques: Expressing

hsa-miR-9-5p down regulates HK2 expression. (A) The results of the dual luciferase reporter gene system showed that hsa-miR-9-5p directly targeted to HK2. (B) The effects of hsa-miR-9-5p on HK2 mRNA was determined using qRT-PCR. HK2 mRNA was significantly reduced by treatment with miR-9-5p agomir. (C) The effects of hsa-miR-9-5p on HK2 protein expression was determined using Western blot. HK2 protein was significantly inhibited by miR-9-5p agomir (** p < 0.01 vs. miR-NC).

Journal: Technology in Cancer Research & Treatment

Article Title: hsa-miR-9-5p Down-Regulates HK2 and Confers Radiosensitivity to Nasopharyngeal Carcinoma

doi: 10.1177/1533033821997822

Figure Lengend Snippet: hsa-miR-9-5p down regulates HK2 expression. (A) The results of the dual luciferase reporter gene system showed that hsa-miR-9-5p directly targeted to HK2. (B) The effects of hsa-miR-9-5p on HK2 mRNA was determined using qRT-PCR. HK2 mRNA was significantly reduced by treatment with miR-9-5p agomir. (C) The effects of hsa-miR-9-5p on HK2 protein expression was determined using Western blot. HK2 protein was significantly inhibited by miR-9-5p agomir (** p < 0.01 vs. miR-NC).

Techniques: Expressing, Luciferase, Quantitative RT-PCR, Western Blot

a Kaplan–Meier estimates of the survival of HCC patients depending on the expression of HK1 , HK2 , HK3 and GCK ( HK4 ) genes in tumor biopsies ( n = 365; diploid samples; TCGA expression data retrieved from cBioPortal; Firehose Legacy) , . Duplicate analyses from the same patient were removed as well as patients who died when biopsied (overall survival=0 months or not specified). Optimal stratification based on highest and lowest gene expression values was determined using Protein Atlas database . b Same as above but patients were stratified based on the GCK/HK2 gene expression ratio. The stratification showing the lowest p value when comparing subgroups of patients with the highest to the lowest GCK/HK2 expression ratio is displayed. Patient TCGA-DD-AAE9 exhibiting undetectable levels of GCK and HK2 was removed from this analysis as the GCK/HK2 ratio could not be calculated. c Correlations between patient survival, GCK expression and HK2 expression. Spearman’s rank correlation test on the subset 130 patients for whom the period between diagnosis and death is precisely known (uncensored data).

Journal: Communications Biology

Article Title: A hexokinase isoenzyme switch in human liver cancer cells promotes lipogenesis and enhances innate immunity

doi: 10.1038/s42003-021-01749-3

Figure Lengend Snippet: a Kaplan–Meier estimates of the survival of HCC patients depending on the expression of HK1 , HK2 , HK3 and GCK ( HK4 ) genes in tumor biopsies ( n = 365; diploid samples; TCGA expression data retrieved from cBioPortal; Firehose Legacy) , . Duplicate analyses from the same patient were removed as well as patients who died when biopsied (overall survival=0 months or not specified). Optimal stratification based on highest and lowest gene expression values was determined using Protein Atlas database . b Same as above but patients were stratified based on the GCK/HK2 gene expression ratio. The stratification showing the lowest p value when comparing subgroups of patients with the highest to the lowest GCK/HK2 expression ratio is displayed. Patient TCGA-DD-AAE9 exhibiting undetectable levels of GCK and HK2 was removed from this analysis as the GCK/HK2 ratio could not be calculated. c Correlations between patient survival, GCK expression and HK2 expression. Spearman’s rank correlation test on the subset 130 patients for whom the period between diagnosis and death is precisely known (uncensored data).

Article Snippet: Primary antibodies used for immunoblotting included mouse monoclonal antibody against human GCK (clone G-6, Santa Cruz Biotechnology), rabbit monoclonal antibody against human HK2 (Clone C64G5, Cell Signaling Technology), rabbit monoclonal antibody against human HK1 (C35C4, Cell Signaling), rabbit polyclonal antibody against human HK3 (HPA056743, Millipore Sigma-Aldrich), goat polyclonal antibody against human ACLY (SAB2500845, Millipore Sigma-Aldrich), rabbit polyclonal antibody against human pACLY (phospho S455, Cell Signaling Technology), rabbit monoclonal antibody against human PDH α1 subunit (C54G1, Cell Signaling Technology), rabbit monoclonal antibody against human pPDH E1-alpha subunit (phospho S293, Abcam), goat polyclonal antibody against human PC (SAB2500845, Millipore Sigma-Aldrich), rabbit monoclonal antibody against human GAPDH (D16H11, Cell Signaling Technology) and rabbit polyclonal antibody against human HIF-1α (NB100-134, Novus Biologicals; 1:500 dilution).

Techniques: Expressing, Gene Expression, Biomarker Discovery

a Western-blot analysis of HK1, HK2, HK3 and GCK expression in Huh7 and Huh7- GCK + /HK2 − . b Hexokinase activity in homogenates of Huh7 and Huh7- GCK + /HK2 − cells. Means ± SEM are presented ( n = 3). c Number of genes changing their expression pattern in Huh7 and Huh7- GCK + /HK2 − cells (see Supplementary Data for details). d Heatmap showing clustering enrichment scores of the networks obtained when mapping differentially expressed genes to the human metabolic model Recon2. Clustering enrichment scores from the highest in red to the lowest in blue were calculated for different gene expression thresholds (Log | FC | ) and percentages of retained currency metabolites. e Gene network corresponding to the maximal clustering enrichment score (Log | FC | > 3; removed currency metabolites = 2%). The transcription of nodes in green was upregulated and those in red downregulated in Huh7- GCK + /HK2 − compared to Huh7 cells. Plain edges mark co-regulation between nodes and broken edges inverse regulation at the transcriptional level.

Journal: Communications Biology

Article Title: A hexokinase isoenzyme switch in human liver cancer cells promotes lipogenesis and enhances innate immunity

doi: 10.1038/s42003-021-01749-3

Figure Lengend Snippet: a Western-blot analysis of HK1, HK2, HK3 and GCK expression in Huh7 and Huh7- GCK + /HK2 − . b Hexokinase activity in homogenates of Huh7 and Huh7- GCK + /HK2 − cells. Means ± SEM are presented ( n = 3). c Number of genes changing their expression pattern in Huh7 and Huh7- GCK + /HK2 − cells (see Supplementary Data for details). d Heatmap showing clustering enrichment scores of the networks obtained when mapping differentially expressed genes to the human metabolic model Recon2. Clustering enrichment scores from the highest in red to the lowest in blue were calculated for different gene expression thresholds (Log | FC | ) and percentages of retained currency metabolites. e Gene network corresponding to the maximal clustering enrichment score (Log | FC | > 3; removed currency metabolites = 2%). The transcription of nodes in green was upregulated and those in red downregulated in Huh7- GCK + /HK2 − compared to Huh7 cells. Plain edges mark co-regulation between nodes and broken edges inverse regulation at the transcriptional level.

Techniques: Western Blot, Expressing, Activity Assay, Gene Expression

Analysis of differentially expressed genes in Huh7 and Huh7- GCK + /HK2 − using gene set enrichment analysis (| FC | > 2 with a p value <0.05).

Journal: Communications Biology

Article Title: A hexokinase isoenzyme switch in human liver cancer cells promotes lipogenesis and enhances innate immunity

doi: 10.1038/s42003-021-01749-3

Figure Lengend Snippet: Analysis of differentially expressed genes in Huh7 and Huh7- GCK + /HK2 − using gene set enrichment analysis (| FC | > 2 with a p value <0.05).

Techniques: Cell Function Assay

$a Quantification of intracellular lipids in total cell extracts of Huh7 and Huh7- GCK + /HK2 − cells ( n = 3). b Lipids and ApoB secretions in supernatants of cells cultured 24 h without FCS ( n = 6 for Cholesterol, n = 3 for FFA and n = 10 for TG and ApoB). c TG/ApoB molar ratio calculated from quantifications determined in b ( n = 10). d Supernatants of Huh7 and Huh7- GCK + /HK2 − were analyzed by ultracentrifugation on iodixanol density gradients. ApoB was quantified in each fraction by ELISA (one representative experiment). Presented data correspond to means ± SEM of indicated number of independent experiments and p values were determined by Student’s t-test.$

Journal: Communications Biology

Article Title: A hexokinase isoenzyme switch in human liver cancer cells promotes lipogenesis and enhances innate immunity

doi: 10.1038/s42003-021-01749-3

Figure Lengend Snippet: a Quantification of intracellular lipids in total cell extracts of Huh7 and Huh7- GCK + /HK2 − cells ( n = 3). b Lipids and ApoB secretions in supernatants of cells cultured 24 h without FCS ( n = 6 for Cholesterol, n = 3 for FFA and n = 10 for TG and ApoB). c TG/ApoB molar ratio calculated from quantifications determined in b ( n = 10). d Supernatants of Huh7 and Huh7- GCK + /HK2 − were analyzed by ultracentrifugation on iodixanol density gradients. ApoB was quantified in each fraction by ELISA (one representative experiment). Presented data correspond to means ± SEM of indicated number of independent experiments and p values were determined by Student’s t-test.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

a Glycogen quantification. b Creatinine and creatinine-P quantification. c This bubble chart compares intracellular metabolomes of Huh7 and Huh7- GCK + /HK2 − cells. Metabolite pool sizes larger in Huh7 are indicated in blue, whereas the one larger in Huh7- GCK + /HK2 − are shown in red. The size of bubbles inversely scales with p values between 5.10 − 2 and 1.10 − 17 of differential metabolomics responses. d Metabolic fluxes for overall glucose consumption and lactate secretion by Huh7 and Huh7- GCK + /HK2 − cells. Indicated values correspond to differences in glucose or lactate concentrations in extracellular culture medium before and after 24 h of culture. e Mass isotopomer distribution vector of pyruvate in cells cultured with [U- 13 C]-glucose. Presented data correspond to n = 24 ( c , d ) or n = 16 ( e ) acquired spectra from N = 6 and N = 4 independent specimens, respectively. f Pyruvate carboxylase (PC) activity determined in cell homogenates. g Western-blot analysis of PC expression in Huh7 and Huh7- GCK + /HK2 − cells. h Western-blot analysis of pyruvate dehydrogenase (PDH) E1-alpha subunit phosphorylation at Ser293. i RNA-seq quantification of pyruvate dehydrogenase kinase 2 (PDK2) and pyruvate dehydrogenase phosphatase 2 (PDP2) (BH adjusted p value<0.05 from transcriptomic data). j Western-blot analysis of ATP-citrate Lyase (ACLY) phosphorylation at Ser455. k Succinate quantification in cell homogenates. l Succinate dehydrogenase (SDH) activity determined in cell homogenates. m Oxygen consumption rate (OCR) in Huh7 and Huh7- GCK + /HK2 − cells was determined with a Seahorse analyzer before and after the addition of oligomycin (Complex V inhibitor), FCCP (uncoupling agent), rotenone (Complex I inhibitor) and antimycin A (Complex III inhibitor) ( n = 5). n Non-mitochondrial, complex I-dependent and complex III-dependent maximal OCR were calculated from m. Except otherwise indicated, data correspond to means ± SEM of 3 independent experiments and p values were determined by Student’s t -test.

Journal: Communications Biology

Article Title: A hexokinase isoenzyme switch in human liver cancer cells promotes lipogenesis and enhances innate immunity

doi: 10.1038/s42003-021-01749-3

Figure Lengend Snippet: a Glycogen quantification. b Creatinine and creatinine-P quantification. c This bubble chart compares intracellular metabolomes of Huh7 and Huh7- GCK + /HK2 − cells. Metabolite pool sizes larger in Huh7 are indicated in blue, whereas the one larger in Huh7- GCK + /HK2 − are shown in red. The size of bubbles inversely scales with p values between 5.10 − 2 and 1.10 − 17 of differential metabolomics responses. d Metabolic fluxes for overall glucose consumption and lactate secretion by Huh7 and Huh7- GCK + /HK2 − cells. Indicated values correspond to differences in glucose or lactate concentrations in extracellular culture medium before and after 24 h of culture. e Mass isotopomer distribution vector of pyruvate in cells cultured with [U- 13 C]-glucose. Presented data correspond to n = 24 ( c , d ) or n = 16 ( e ) acquired spectra from N = 6 and N = 4 independent specimens, respectively. f Pyruvate carboxylase (PC) activity determined in cell homogenates. g Western-blot analysis of PC expression in Huh7 and Huh7- GCK + /HK2 − cells. h Western-blot analysis of pyruvate dehydrogenase (PDH) E1-alpha subunit phosphorylation at Ser293. i RNA-seq quantification of pyruvate dehydrogenase kinase 2 (PDK2) and pyruvate dehydrogenase phosphatase 2 (PDP2) (BH adjusted p value<0.05 from transcriptomic data). j Western-blot analysis of ATP-citrate Lyase (ACLY) phosphorylation at Ser455. k Succinate quantification in cell homogenates. l Succinate dehydrogenase (SDH) activity determined in cell homogenates. m Oxygen consumption rate (OCR) in Huh7 and Huh7- GCK + /HK2 − cells was determined with a Seahorse analyzer before and after the addition of oligomycin (Complex V inhibitor), FCCP (uncoupling agent), rotenone (Complex I inhibitor) and antimycin A (Complex III inhibitor) ( n = 5). n Non-mitochondrial, complex I-dependent and complex III-dependent maximal OCR were calculated from m. Except otherwise indicated, data correspond to means ± SEM of 3 independent experiments and p values were determined by Student’s t -test.

Techniques: Plasmid Preparation, Cell Culture, Activity Assay, Western Blot, Expressing, Phospho-proteomics, RNA Sequencing

a Sector chart from the transcriptomic study showing genes included in the GO-term “Type I-IFN signaling pathway”. b List of genes significantly up-regulated in red or down-regulated in purple (| FC | > 2, BH adjusted p value<0.05) in Huh7- GCK + /HK2 − compared to Huh7 cells ( n = 3). c – e Cells were stimulated or not for 48 h with 3p-hpRNA (RIG-I ligand) or poly(I:C) (IFIH1/MDA5 ligand). ISRE-luciferase expression was monitored and normalized to Renilla luciferase ( c , d ) ( n = 3 for 3p-hpRNA and n = 4 for poly(I:C) treatments). Cell supernatants were assayed for cytokine concentration by multiplex assays ( n = 3 to 7) ( e ). f NK cell mediated lysis of Huh7 or Huh7- GCK + /HK2 − cells. Hepatoma cells were seeded 24 h before NK cells addition for 4 h at effector to target (E:T) ratio of 0, 3 or 30. After harvesting, cell lysis was determined by the percentage of PI + cells on gated hepatocytes ( n = 3). Means ± SEM of indicated n independent experiments are presented and p values were obtained from 2-way ANOVA analyses comparing matched cell means with Sidak’s correction for multiple comparison, with α = 0.05.

Journal: Communications Biology

Article Title: A hexokinase isoenzyme switch in human liver cancer cells promotes lipogenesis and enhances innate immunity

doi: 10.1038/s42003-021-01749-3

Figure Lengend Snippet: a Sector chart from the transcriptomic study showing genes included in the GO-term “Type I-IFN signaling pathway”. b List of genes significantly up-regulated in red or down-regulated in purple (| FC | > 2, BH adjusted p value<0.05) in Huh7- GCK + /HK2 − compared to Huh7 cells ( n = 3). c – e Cells were stimulated or not for 48 h with 3p-hpRNA (RIG-I ligand) or poly(I:C) (IFIH1/MDA5 ligand). ISRE-luciferase expression was monitored and normalized to Renilla luciferase ( c , d ) ( n = 3 for 3p-hpRNA and n = 4 for poly(I:C) treatments). Cell supernatants were assayed for cytokine concentration by multiplex assays ( n = 3 to 7) ( e ). f NK cell mediated lysis of Huh7 or Huh7- GCK + /HK2 − cells. Hepatoma cells were seeded 24 h before NK cells addition for 4 h at effector to target (E:T) ratio of 0, 3 or 30. After harvesting, cell lysis was determined by the percentage of PI + cells on gated hepatocytes ( n = 3). Means ± SEM of indicated n independent experiments are presented and p values were obtained from 2-way ANOVA analyses comparing matched cell means with Sidak’s correction for multiple comparison, with α = 0.05.

Techniques: Luciferase, Expressing, Concentration Assay, Multiplex Assay, Lysis, Comparison

The relationship between PVT1 expression status and clinic-pathologic features of gallbladder cancer

Journal: Molecular Cancer

Article Title: Long non-coding RNA PVT1 promotes tumor progression by regulating the miR-143/HK2 axis in gallbladder cancer

doi: 10.1186/s12943-019-0947-9

Figure Lengend Snippet: The relationship between PVT1 expression status and clinic-pathologic features of gallbladder cancer

Article Snippet: The slides were incubated with rabbit anti-human polyclonal antibodies against HK2 (1:200 dilution, Proteintech) and Ki-67 (1:500 dilution, Signalway Antibody) at 4 °C overnight and then probed with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, CA, USA) and high-sensitivity streptavidin–HRP conjugate.

Techniques: Expressing

Univariate and multivariate analyses of overall survival of gallbladder cancer

Journal: Molecular Cancer

Article Title: Long non-coding RNA PVT1 promotes tumor progression by regulating the miR-143/HK2 axis in gallbladder cancer

doi: 10.1186/s12943-019-0947-9

Figure Lengend Snippet: Univariate and multivariate analyses of overall survival of gallbladder cancer

Techniques: Expressing

PVT1 regulates HK2 expression by sponging miR-143. (a-b) The effects of si-PVT1, miR-143 inhibitor and si-PVT1 + miR-143 inhibitor on mRNA and protein levels of HK2 in GBC-SD cells. (c-d) The effects of miR-143 mimics, pcDNA-PVT1, and miR-143 mimics+pcDNA-PVT1 on mRNA and protein levels of HK2 in GBC-SD cells. (e-f) Pearson correlation between PVT1 and HK2 mRNA transcript level was measured in GSE76633 and 20 pairs of GBC tissues, respectively. (g-h) Expression level of HK2 was higher in GBC tissues than in normal tissues by IHC staining. Scale bar, 100 μm. (i) Representative HK2 staining patterns. Scale bar, 100 μm. (j-k) High PVT1 expression correlated with advanced TNM stage, metastasis and poor overall survival. (l) The association between PVT1 and HK2 expression. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate mean ± SD

Journal: Molecular Cancer

Article Title: Long non-coding RNA PVT1 promotes tumor progression by regulating the miR-143/HK2 axis in gallbladder cancer

doi: 10.1186/s12943-019-0947-9

Figure Lengend Snippet: PVT1 regulates HK2 expression by sponging miR-143. (a-b) The effects of si-PVT1, miR-143 inhibitor and si-PVT1 + miR-143 inhibitor on mRNA and protein levels of HK2 in GBC-SD cells. (c-d) The effects of miR-143 mimics, pcDNA-PVT1, and miR-143 mimics+pcDNA-PVT1 on mRNA and protein levels of HK2 in GBC-SD cells. (e-f) Pearson correlation between PVT1 and HK2 mRNA transcript level was measured in GSE76633 and 20 pairs of GBC tissues, respectively. (g-h) Expression level of HK2 was higher in GBC tissues than in normal tissues by IHC staining. Scale bar, 100 μm. (i) Representative HK2 staining patterns. Scale bar, 100 μm. (j-k) High PVT1 expression correlated with advanced TNM stage, metastasis and poor overall survival. (l) The association between PVT1 and HK2 expression. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate mean ± SD

Techniques: Expressing, Immunohistochemistry, Staining

HK2 promotes cell proliferation, invasion and migration in vitro and tumor growth in vivo. (a) The protein levels of HK2 in GBC cells after knockdown by si-HK2. (b-e) Knockdown of HK2 significantly decreased cell proliferation compared with si-ctrl cells by proliferation assays. (f) Formation of spheres from GBC cells transfected with si-HK2 accessed by three-dimensional cell culture (magnification, × 200, scale bar, 50 μm). (g-i) HK2 suppression impaired GBC cell invasion and migration, as measured by transwell assay (magnification, × 100). Scale bar, 100 μm and wound healing assays. (j-k) Glucose consumption and lactate production were significantly decreased after HK2 knockdown. (l-m) Tumor volume and weight in the lenti-sh-HK2 group were significantly lower than those in the lenti-sh-NC group. (n) Images of tumor formation were performed by a live imaging system detecting the luciferase signal. (o) The luciferase activity in the lenti-sh-HK2 group was lower than that in the lenti-sh-NC group. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate mean ± SD

Journal: Molecular Cancer

Article Title: Long non-coding RNA PVT1 promotes tumor progression by regulating the miR-143/HK2 axis in gallbladder cancer

doi: 10.1186/s12943-019-0947-9

Figure Lengend Snippet: HK2 promotes cell proliferation, invasion and migration in vitro and tumor growth in vivo. (a) The protein levels of HK2 in GBC cells after knockdown by si-HK2. (b-e) Knockdown of HK2 significantly decreased cell proliferation compared with si-ctrl cells by proliferation assays. (f) Formation of spheres from GBC cells transfected with si-HK2 accessed by three-dimensional cell culture (magnification, × 200, scale bar, 50 μm). (g-i) HK2 suppression impaired GBC cell invasion and migration, as measured by transwell assay (magnification, × 100). Scale bar, 100 μm and wound healing assays. (j-k) Glucose consumption and lactate production were significantly decreased after HK2 knockdown. (l-m) Tumor volume and weight in the lenti-sh-HK2 group were significantly lower than those in the lenti-sh-NC group. (n) Images of tumor formation were performed by a live imaging system detecting the luciferase signal. (o) The luciferase activity in the lenti-sh-HK2 group was lower than that in the lenti-sh-NC group. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate mean ± SD

Techniques: Migration, In Vitro, In Vivo, Knockdown, Transfection, Cell Culture, Transwell Assay, Imaging, Luciferase, Activity Assay

The PVT1/miR-143/HK2 axis regulates glucose metabolism in GBC cells. (a-b) The effects of si-PVT1, miR-143 inhibitor and si-PVT1 + miR-143 inhibitor on glucose consumption and cellular lactate production levels in GBC-SD cells. (c-d) The effects of miR-143 mimics, pcDNA-PVT1, and miR-143 mimics+pcDNA-PVT1 on glucose consumption and cellular lactate production levels in GBC-SD cells. (e-f) ECAR was detected by the Glycolysis Stress Test in GBC-SD cells after transfection with si-PVT1 or si-NC. (g-h) OCR was measured by the Cell Mito Stress Test in GBC-SD cells after transfection with si-PVT1 or si-NC. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate mean ± SD

Journal: Molecular Cancer

Article Title: Long non-coding RNA PVT1 promotes tumor progression by regulating the miR-143/HK2 axis in gallbladder cancer

doi: 10.1186/s12943-019-0947-9

Figure Lengend Snippet: The PVT1/miR-143/HK2 axis regulates glucose metabolism in GBC cells. (a-b) The effects of si-PVT1, miR-143 inhibitor and si-PVT1 + miR-143 inhibitor on glucose consumption and cellular lactate production levels in GBC-SD cells. (c-d) The effects of miR-143 mimics, pcDNA-PVT1, and miR-143 mimics+pcDNA-PVT1 on glucose consumption and cellular lactate production levels in GBC-SD cells. (e-f) ECAR was detected by the Glycolysis Stress Test in GBC-SD cells after transfection with si-PVT1 or si-NC. (g-h) OCR was measured by the Cell Mito Stress Test in GBC-SD cells after transfection with si-PVT1 or si-NC. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate mean ± SD

Techniques: Transfection

PVT1 promotes tumor growth in vivo. (a) Images of tumor formation were performed by a live imaging system detecting the luciferase signal. (b) The luciferase activity in the lenti-sh-PVT1 group was lower than in the lenti-sh-NC group. (c-d) The volumes and weight of lenti-sh-PVT1 cell-derived xenograft tumors were markedly lower than those of the lenti-sh-NC group. (e-g) Sections of xenograft tumors stained with hematoxylin and eosin (H&E) as well as immunohistochemical staining for HK2 and Ki-67 (magnification, × 200). Scale bar, 100 μm. (h) Representative images of tumor formation of the lenti-NC group and lenti-PVT1 group. (i) The luciferase activity in the lenti-PVT1 group was higher than that in the lenti-NC group. (j-k) The volumes and weight of lenti-PVT1 cell-derived xenograft tumors were markedly higher than those of the lenti-NC group. (l-n) The levels of Ki67 and HK2 were much higher after PVT1 overexpression (magnification, × 200). Scale bar, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars indicate mean ± SD

Journal: Molecular Cancer

Article Title: Long non-coding RNA PVT1 promotes tumor progression by regulating the miR-143/HK2 axis in gallbladder cancer

doi: 10.1186/s12943-019-0947-9

Figure Lengend Snippet: PVT1 promotes tumor growth in vivo. (a) Images of tumor formation were performed by a live imaging system detecting the luciferase signal. (b) The luciferase activity in the lenti-sh-PVT1 group was lower than in the lenti-sh-NC group. (c-d) The volumes and weight of lenti-sh-PVT1 cell-derived xenograft tumors were markedly lower than those of the lenti-sh-NC group. (e-g) Sections of xenograft tumors stained with hematoxylin and eosin (H&E) as well as immunohistochemical staining for HK2 and Ki-67 (magnification, × 200). Scale bar, 100 μm. (h) Representative images of tumor formation of the lenti-NC group and lenti-PVT1 group. (i) The luciferase activity in the lenti-PVT1 group was higher than that in the lenti-NC group. (j-k) The volumes and weight of lenti-PVT1 cell-derived xenograft tumors were markedly higher than those of the lenti-NC group. (l-n) The levels of Ki67 and HK2 were much higher after PVT1 overexpression (magnification, × 200). Scale bar, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars indicate mean ± SD

Techniques: In Vivo, Imaging, Luciferase, Activity Assay, Derivative Assay, Staining, Immunohistochemical staining, Over Expression

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