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mouse anti human hk2 (Santa Cruz Biotechnology)

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Santa Cruz Biotechnology mouse anti human hk2

FIGURE 1 <t>HK2</t> translocates to mitochondria after CoCl2 and PDGF stimulation. (A) Confocal microscopy of RA FLS (n = 3) after stimulation with 150 µM cobalt chloride (CoCl2) 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.

Mouse Anti Human Hk2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes."

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

Journal: Frontiers in immunology

doi: 10.3389/fimmu.2023.1103231

FIGURE 1 HK2 translocates to mitochondria after CoCl2 and PDGF stimulation. (A) Confocal microscopy of RA FLS (n = 3) after stimulation with 150 µM cobalt chloride (CoCl2) 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.

Figure Legend Snippet: FIGURE 1 HK2 translocates to mitochondria after CoCl2 and PDGF stimulation. (A) Confocal microscopy of RA FLS (n = 3) after stimulation with 150 µM cobalt chloride (CoCl2) 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.

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

FIGURE 2 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- HK2DN 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.

Figure Legend Snippet: FIGURE 2 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- HK2DN 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 Used: Mutagenesis, Migration, Infection, Control, Construct, Comparison, Sterility, Transferring

FIGURE 3 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 H2O2, 75 µM H2O2, and 200 µM H2O2 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.

Figure Legend Snippet: FIGURE 3 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 H2O2, 75 µM H2O2, and 200 µM H2O2 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 Used: Migration, Comparison, Sterility, Transferring, Staining, Software, Control

FIGURE 4 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 CoCl2 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.

Figure Legend Snippet: FIGURE 4 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 CoCl2 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 Used: Translocation Assay, Confocal Microscopy, Staining, Comparison

FIGURE 5 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.

Figure Legend Snippet: FIGURE 5 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 Used: Expressing, Gene Expression, Marker

FIGURE 6 Adenovirus injection and Ad-15G treatment in AIA model. (A, B) B6 mice (n = 10) injected with AdenoHK2FL or Adeno HK2DN in either knee as described in methods. (A) Histological scores of mouse knees measuring synovial inﬁltration and inﬁltration of either HK2FL or HK2DN. 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.

Figure Legend Snippet: FIGURE 6 Adenovirus injection and Ad-15G treatment in AIA model. (A, B) B6 mice (n = 10) injected with AdenoHK2FL or Adeno HK2DN in either knee as described in methods. (A) Histological scores of mouse knees measuring synovial inﬁltration and inﬁltration of either HK2FL or HK2DN. 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 Used: Injection, Comparison, Two Tailed Test, MANN-WHITNEY, Control

Image Search Results

a , b The expression of glycolysis-related genes was detected by RT-qPCR in both B7-H3-overexpressing HCT116 ( a ) and RKO ( b ) cells. c , d The mRNA level of HK2 was detected by RT-qPCR in both HCT116 ( c ) and RKO ( d ) cells after transfection with siRNA NC, B7-H3 siRNA-1 or B7-H3 siRNA-2. e HK2 protein level and STAT3 activation (examined by the p-STAT3 expression level) were detected by western blot in both HCT116 and RKO cells after transfection with NC, B7-H3 siRNA-1 or B7-H3 siRNA-2. β-actin served as a loading control. f HK2 protein level and STAT3 activation (examined by the p-STAT3 expression level) were detected by western blot in both B7-H3-overexpressing HCT116 and RKO cells. β-actin served as a loading control. g Schematic representation of the proposed B7-H3/STAT3/HK2 axis. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a , b The expression of glycolysis-related genes was detected by RT-qPCR in both B7-H3-overexpressing HCT116 ( a ) and RKO ( b ) cells. c , d The mRNA level of HK2 was detected by RT-qPCR in both HCT116 ( c ) and RKO ( d ) cells after transfection with siRNA NC, B7-H3 siRNA-1 or B7-H3 siRNA-2. e HK2 protein level and STAT3 activation (examined by the p-STAT3 expression level) were detected by western blot in both HCT116 and RKO cells after transfection with NC, B7-H3 siRNA-1 or B7-H3 siRNA-2. β-actin served as a loading control. f HK2 protein level and STAT3 activation (examined by the p-STAT3 expression level) were detected by western blot in both B7-H3-overexpressing HCT116 and RKO cells. β-actin served as a loading control. g Schematic representation of the proposed B7-H3/STAT3/HK2 axis. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Article Snippet: The antibodies for the western blot analysis in this study were as follows: goat anti-human 4IgB7-H3 (R&D Systems, #AF1027), mouse anti-human HK2 (Novus Biologicals, #NBP1-51643), rabbit anti-human/mouse STAT3 (CST, #12640, MA, USA), rabbit anti-human/mouse Phospho-STAT3 (pSTAT3) (CST, #9145), rabbit anti-human Bcl-2 (abcam, #ab32124), rabbit anti-human/mouse Bax (abcam, #ab32503) and mouse anti-human/mouse β-actin (CST, #3700).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Activation Assay, Western Blot, Control

a The protein level of HK2 in B7-H3-overexpressing HCT116 or RKO cells after transfection with siRNA negative control (NC) or HK2 siRNA transfection were analyzed by western blot. β-actin served as a loading control. b , c Glucose consumption ( b ) and lactate production ( c ) were measured in B7-H3-overexpressing HCT116 or RKO cells transfected with NC or HK2 siRNA. d , e Glucose consumption ( d ) and lactate production ( e ) were measured in B7-H3-overexpressing HCT116 or RKO cells treated with PBS or 2-DG. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a The protein level of HK2 in B7-H3-overexpressing HCT116 or RKO cells after transfection with siRNA negative control (NC) or HK2 siRNA transfection were analyzed by western blot. β-actin served as a loading control. b , c Glucose consumption ( b ) and lactate production ( c ) were measured in B7-H3-overexpressing HCT116 or RKO cells transfected with NC or HK2 siRNA. d , e Glucose consumption ( d ) and lactate production ( e ) were measured in B7-H3-overexpressing HCT116 or RKO cells treated with PBS or 2-DG. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Techniques: Transfection, Negative Control, Western Blot, Control

a , b Colony formation assay of B7-H3-overexpressing HCT116 or RKO cells exposed to L-OHP. The chemoresistance of B7-H3 was abolished by HK2 siRNA or 2-DG. c , d Cell viability was analyzed using the CCK-8 kit. B7-H3-overexpressing HCT116 or RKO cells showed resistance to L-OHP ( c ) or 5-FU ( d ). The chemoresistance of B7-H3 was abolished by HK2 siRNA or 2-DG. e B7-H3-overexpressing HCT116 or RKO cells showed less cell apoptosis than control cells (EV) after L-OHP treatment. The effect of B7-H3 on cell apoptosis was abolished by HK2 siRNA or 2-DG. f The protein levels of Bcl-2 and Bax were detected by western blot in both B7-H3-overexpressing HCT116 and RKO cells. The effect of B7-H3 on Bcl-2 and Bax expression was abolished by HK2 siRNA. β-actin served as a loading control. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a , b Colony formation assay of B7-H3-overexpressing HCT116 or RKO cells exposed to L-OHP. The chemoresistance of B7-H3 was abolished by HK2 siRNA or 2-DG. c , d Cell viability was analyzed using the CCK-8 kit. B7-H3-overexpressing HCT116 or RKO cells showed resistance to L-OHP ( c ) or 5-FU ( d ). The chemoresistance of B7-H3 was abolished by HK2 siRNA or 2-DG. e B7-H3-overexpressing HCT116 or RKO cells showed less cell apoptosis than control cells (EV) after L-OHP treatment. The effect of B7-H3 on cell apoptosis was abolished by HK2 siRNA or 2-DG. f The protein levels of Bcl-2 and Bax were detected by western blot in both B7-H3-overexpressing HCT116 and RKO cells. The effect of B7-H3 on Bcl-2 and Bax expression was abolished by HK2 siRNA. β-actin served as a loading control. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Techniques: Colony Assay, CCK-8 Assay, Control, Western Blot, Expressing

a Images of IHC analysis of B7-H3 and HK2 protein expression and hematoxylin and eosin (H&E) staining of CRC ( n = 126) tissue sections. One representative image is shown. b , c B7-H3 ( b ) and HK2 ( c ) protein expression based on their staining index in nonmalignant adjacent tissues (NAT) and CRC specimens. d Correlation analysis of the staining index of expression levels of B7-H3 and HK2 protein in human CRC specimens ( n = 126). e , f B7-H3 ( e ) and HK2 ( f ) protein expression based on their staining index in CRC specimens at different clinical stages. Values are expressed as means (SEMs). * P < 0.05

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a Images of IHC analysis of B7-H3 and HK2 protein expression and hematoxylin and eosin (H&E) staining of CRC ( n = 126) tissue sections. One representative image is shown. b , c B7-H3 ( b ) and HK2 ( c ) protein expression based on their staining index in nonmalignant adjacent tissues (NAT) and CRC specimens. d Correlation analysis of the staining index of expression levels of B7-H3 and HK2 protein in human CRC specimens ( n = 126). e , f B7-H3 ( e ) and HK2 ( f ) protein expression based on their staining index in CRC specimens at different clinical stages. Values are expressed as means (SEMs). * P < 0.05

Techniques: Expressing, Staining

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: FIGURE 1 HK2 translocates to mitochondria after CoCl2 and PDGF stimulation. (A) Confocal microscopy of RA FLS (n = 3) after stimulation with 150 µM cobalt chloride (CoCl2) 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 frontiersin.org (#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, Control, Comparison, Two Tailed Test

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: FIGURE 2 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- HK2DN 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

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: FIGURE 3 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 H2O2, 75 µM H2O2, and 200 µM H2O2 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

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: FIGURE 4 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 CoCl2 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, Comparison

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: FIGURE 5 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

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: FIGURE 6 Adenovirus injection and Ad-15G treatment in AIA model. (A, B) B6 mice (n = 10) injected with AdenoHK2FL or Adeno HK2DN in either knee as described in methods. (A) Histological scores of mouse knees measuring synovial inﬁltration and inﬁltration of either HK2FL or HK2DN. 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

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

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Article Snippet: Briefly, sections from paraffin embedded tissues were incubated with goat anti-human 4IgB7-H3 antibody (R&D Systems, #AF1027) or mouse anti-human HK2 antibody (Novus Biologicals, #NBP1-51643) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Activation Assay, Western Blot, Control

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Techniques: Transfection, Negative Control, Western Blot, Control

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Techniques: Colony Assay, CCK-8 Assay, Control, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Techniques: Expressing, Staining

Figure 2. Expression levels of glycolysis regulation proteins in SW480 and SW620 cells with different metabolic types. (A and B) Representative western blotting images of HIF-1α, HK1, HK2 and GLUT1 protein expressions in (A) SW480 and (B) SW620 cells cultured in different media. (C-F) Protein expression levels from (A) and (B) were quantified using Image Lab analysis software; the relative expression levels of HIF-1α, HK1, HK2 and GLUT1 were calculated as a ratio of OXPHOS; β-actin was used as the loading control. (G) Western blot analysis of HK1 and HK2 protein expression levels in SW480 and SW620 cells treated with Oligomycin A (1 µM) for 0-48 h. (G) Representative western blots indicating the expression of HK1 and HK2 in SW480 and SW620 cells at different treatment times. (H and I) Protein expression levels from (G) were quantified using Image Lab analysis software, and relative expression levels of (H) HK1 and (I) HK2 were calculated as a ratio of 0 h; β-actin was used as a loading control. Data are presented as the mean ± standard deviation; n=3; **P<0.01 and ***P<0.001 vs. SW480. GLUT1, glucose transporter type 1; HIF-1α, hypoxia-inducible factor; HK, hexokinase; NOR, normal; GLY, glycolysis; OXPHOS, oxidative phosphorylation.

Journal: International journal of oncology

Article Title: Metastatic cancer cells compensate for low energy supplies in hostile microenvironments with bioenergetic adaptation and metabolic reprogramming.

doi: 10.3892/ijo.2018.4582

Figure Lengend Snippet: Figure 2. Expression levels of glycolysis regulation proteins in SW480 and SW620 cells with different metabolic types. (A and B) Representative western blotting images of HIF-1α, HK1, HK2 and GLUT1 protein expressions in (A) SW480 and (B) SW620 cells cultured in different media. (C-F) Protein expression levels from (A) and (B) were quantified using Image Lab analysis software; the relative expression levels of HIF-1α, HK1, HK2 and GLUT1 were calculated as a ratio of OXPHOS; β-actin was used as the loading control. (G) Western blot analysis of HK1 and HK2 protein expression levels in SW480 and SW620 cells treated with Oligomycin A (1 µM) for 0-48 h. (G) Representative western blots indicating the expression of HK1 and HK2 in SW480 and SW620 cells at different treatment times. (H and I) Protein expression levels from (G) were quantified using Image Lab analysis software, and relative expression levels of (H) HK1 and (I) HK2 were calculated as a ratio of 0 h; β-actin was used as a loading control. Data are presented as the mean ± standard deviation; n=3; **P<0.01 and ***P<0.001 vs. SW480. GLUT1, glucose transporter type 1; HIF-1α, hypoxia-inducible factor; HK, hexokinase; NOR, normal; GLY, glycolysis; OXPHOS, oxidative phosphorylation.

Article Snippet: Mouse anti-HK1 (cat. no. BM1910) and mouse anti-human HK2 (cat. no. BM1911) antibodies were purchased from Boster Biological Technology (Pleasanton, CA, USA).

Techniques: Expressing, Western Blot, Cell Culture, Software, Control, Standard Deviation, Phospho-proteomics

Figure 6. RT-qPCR analysis of SW480 and SW620 cells in different culture microenvironments. (A-C) RT-qPCR analysis of the relative mRNA expression levels of HK1, HK2, HIF-1α, CDH1, FN1, VIM, CD133, SNAI1, SNAI2, ZEB1, CDH2 and TWIST1 in SW480 and SW620 cells cultured in different media conditions; mRNA expressions were normalized to β-actin. (D) SW620/SW480 mRNA expression ratios in different types of metabolism. Data are presented as the mean ± standard deviation; n=3; *P<0.05, **P<0.01 and ***P<0.001 vs. SW480. CDH1, Epithelial cadherin; FN1, fibronectin; HIF-1α, hypoxia-inducible factor; HK, hexokinase; Oligo A, Oligomycin A; OXPHOS, oxidative phosphorylation; SNAI, snail family transcriptional repressor; VIM, vimentin; ZEB, Zinc-finger E-box-binding homeobox; NOR, normal; GLY, glycolysis; OXPH, oxidative phosphorylation.

Journal: International journal of oncology

Article Title: Metastatic cancer cells compensate for low energy supplies in hostile microenvironments with bioenergetic adaptation and metabolic reprogramming.

doi: 10.3892/ijo.2018.4582

Figure Lengend Snippet: Figure 6. RT-qPCR analysis of SW480 and SW620 cells in different culture microenvironments. (A-C) RT-qPCR analysis of the relative mRNA expression levels of HK1, HK2, HIF-1α, CDH1, FN1, VIM, CD133, SNAI1, SNAI2, ZEB1, CDH2 and TWIST1 in SW480 and SW620 cells cultured in different media conditions; mRNA expressions were normalized to β-actin. (D) SW620/SW480 mRNA expression ratios in different types of metabolism. Data are presented as the mean ± standard deviation; n=3; *P<0.05, **P<0.01 and ***P<0.001 vs. SW480. CDH1, Epithelial cadherin; FN1, fibronectin; HIF-1α, hypoxia-inducible factor; HK, hexokinase; Oligo A, Oligomycin A; OXPHOS, oxidative phosphorylation; SNAI, snail family transcriptional repressor; VIM, vimentin; ZEB, Zinc-finger E-box-binding homeobox; NOR, normal; GLY, glycolysis; OXPH, oxidative phosphorylation.

Article Snippet: Mouse anti-HK1 (cat. no. BM1910) and mouse anti-human HK2 (cat. no. BM1911) antibodies were purchased from Boster Biological Technology (Pleasanton, CA, USA).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Standard Deviation, Phospho-proteomics, Binding Assay

Figure 7. mRNA levels in SW480 and SW620 cells treated with Oligo A over time. Reverse transcription-quantitative polymerase chain reaction analysis of mRNA expression levels of HK1, HK2, HIF-1α, CDH1, CDH2, ZEB1, SNAI1, SNAI2, TWIST1, CD133, VIM and FN in SW480 and SW620 cells treated with Oligomycin A (1 µM) for 0-48 h. mRNA expression levels were normalized to β-actin. Data are presented as the mean ± standard deviation; n=3; **P<0.01; ***P<0.001 vs. SW480. CDH1, epithelial cadherin; CDH2, Neural-cadherin; FN1, fibronectin; HIF-1α, hypoxia-inducible factor; HK, hexokinase; SNAI, snail family transcriptional repressor; VIM, vimentin; ZEB, Zinc-finger E-box-binding homeobox.

Journal: International journal of oncology

Article Title: Metastatic cancer cells compensate for low energy supplies in hostile microenvironments with bioenergetic adaptation and metabolic reprogramming.

doi: 10.3892/ijo.2018.4582

Figure Lengend Snippet: Figure 7. mRNA levels in SW480 and SW620 cells treated with Oligo A over time. Reverse transcription-quantitative polymerase chain reaction analysis of mRNA expression levels of HK1, HK2, HIF-1α, CDH1, CDH2, ZEB1, SNAI1, SNAI2, TWIST1, CD133, VIM and FN in SW480 and SW620 cells treated with Oligomycin A (1 µM) for 0-48 h. mRNA expression levels were normalized to β-actin. Data are presented as the mean ± standard deviation; n=3; **P<0.01; ***P<0.001 vs. SW480. CDH1, epithelial cadherin; CDH2, Neural-cadherin; FN1, fibronectin; HIF-1α, hypoxia-inducible factor; HK, hexokinase; SNAI, snail family transcriptional repressor; VIM, vimentin; ZEB, Zinc-finger E-box-binding homeobox.

Article Snippet: Mouse anti-HK1 (cat. no. BM1910) and mouse anti-human HK2 (cat. no. BM1911) antibodies were purchased from Boster Biological Technology (Pleasanton, CA, USA).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Binding Assay

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