Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A and B. IL-11 mRNA measured with reverse transcription polymerase chain reaction (RTPCR) in HK-2 cells treated with 0-2.5% isoflurane for 6 h (A. N = 6) or 2.5% isoflurane for 0-6 h (B, N = 6). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was quantified to normalize lane loading. Data are presented as means ± SD. * P < 0.05 vs. IL-11 mRNA measured after 0% isoflurane-treatment (A) or at 0 h (B). C. Isoflurane increases IL-11 protein (pg/ml) in cell culture media from HK-2 cells (N = 6). HK-2 cells were treated with 2.5% isoflurane or with carrier gas for 6 h or 16 h. * P < 0.05 vs. carrier gas treated group.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. IL-11 messenger ribonucleic acid (mRNA) (detected with reverse transcription polymerase chain reaction (RTPCR)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas plus immunoglobulin G (IgG) isotype antibody treated controls. B. IL-11 protein (detected with enzyme-linked immunosorbent assay (ELISA)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). * P < 0.05 vs. carrier gas group treated with IgG isotype antibody. # P < 0.05 vs. isoflurane group treated with IgG isotype antibody. Error bars represent 1 SD. TGF-β1 antibody (10 μg/ml) prevents isoflurane-mediated induction of IL-11 mRNA and protein expression in human proximal tubule cells.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: IL-11 mRNA (reverse transcription polymerase chain reaction (RTPCR)) expression in primary culture of mouse proximal tubule cells treated with 2.5% isoflurane for 6 h (N = 4). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas and immunoglobulin G (IgG) isotype antibody treated controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was also quantified to normalize lane loading. * P < 0.05 vs. carrier gas group treated with IgG isotype antibody. # P < 0.05 vs. isoflurane group treated with IgG isotype antibody. Error bars represent 1 SD. TGF-β1 antibody (10 μg/ml) prevents isoflurane-mediated induction of IL-11 mRNA and protein expression in mouse proximal tubule cells.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: Naïve IL-11 wild-type (WT) mice were exposed to pentobarbital (PB) or 1.2% isoflurane (ISO) for 4 h. A. Representative bands for IL-11 mRNA (reverse transcription polymerase chain reaction (RTPCR)) expression in mouse kidney (N = 4). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal loading control. B. Kidney lysate IL-11 protein (detected by enzyme-linked immunosorbent assay (ELISA)) in IL-11 naïve WT mice exposed to pentobarbital (PB) or 1.2% isoflurane (ISO) for 4 h (N = 4). To neutralize TGF-β1 in vivo, some IL-11 WT mice were injected with 5 mg/kg monoclonal anti-TGF-β1 (MAB240) antibody intravenous injection. TGF-β1 neutralization prevented the induction of IL-11 after isoflurane anesthesia. * P < 0.05 vs. pentobarbital anesthetized mice treated with immunoglobulin G (IgG) isotype antibody. # P < 0.05 vs. isoflurane anesthetized mice treated with IgG isotype antibody. Error bars represent 1 SD. Isoflurane anesthesia significantly increased kidney IL-11 mRNA and protein expression in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Enzyme-linked Immunosorbent Assay, In Vivo, Injection, Neutralization

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. IL-11 immunohistochemistry (400× shown, N = 4) in kidneys from IL-11 receptor wild type (IL-11R WT) mice anesthetized with pentobarbital or with 1.2% isoflurane for 4 h. IL-11 staining was darker in kidneys of mice anesthetized with isoflurane. TGF-β1 neutralizing antibody attenuated isoflurane-mediated increase in IL-11 immunoreactivity. Finally, IL-11 staining was not visible in kidneys from mice stained with negative isotype control antibodies (representative of four experiments). B. Quantifications of renal tubular IL-11 staining in mice anesthetized with pentobarbital or with 1.2% isoflurane for 4 h. Kidney IL-11 immunoreactivity significantly increased in mice anesthetized with isoflurane and attenuated with TGF-β1 neutralizing antibody. # P < 0.05 vs. isoflurane anesthetized mice treated with immunoglobulin G (IgG) isotype antibody. * P < 0.05 vs. pentobarbital-anesthetized mice. Error bars represent 1 SD.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Immunohistochemistry, Staining, Control

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: Plasma creatinine levels from IL-11 wild-type (WT) or IL-11 deficient (KO) mice subjected to 30 min renal ischemia and 24 h reperfusion (N = 4-6 per group). After renal ischemia reperfusion (RIR), mice were further anesthetized with 1.2% isoflurane (ISO) or with equi-anesthetic dose of pentobarbital (PB). Some IL-11 receptor (IL-11R) WT mice were pretreated with an IL-11 neutralizing antibody (1 mg/kg, intravenous injection) 20 min before reperfusion or sham-operation. Isoflurane postconditioning significantly reduced plasma creatinine after RIR injury in IL-11R WT mice. However, IL-11R deficiency or IL-11 neutralizing antibody prevented the renal protective effects of isoflurane post-conditioning. * P < 0.05 vs. respective sham-operated mice. # P < 0.05 vs. pentobarbital anesthetized mice subjected to RIR. Data are from 6 mice per group and represented as mean ± SD.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Clinical Proteomics, Injection

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. Representative photomicrographs of five to six experiments for hematoxylin and eosin staining (magnification 200×) of kidneys of IL-11 receptor wild-type (IL-11R WT) mice, IL-11 receptor deficient (IL-11R KO) mice and IL-11R WT mice pretreated with IL-11 neutralizing antibody and subjected to 30 min renal ischemia and 24-h reperfusion (I/R). B. Summary of Jablonski scale renal injury scores (N = 4, graded from hematoxylin and eosin staining, scale 0-4) for mice subjected to renal I/R. * P < 0.05 vs. pentobarbital-anesthetized IL-11R WT mice subjected to renal I/R. Error bars represent 1 SD. IL-11R WT mice anesthetized with pentobarbital after renal ischemia showed severe renal tubular necrosis. Isoflurane post-conditioning significantly attenuated renal tubular necrosis and renal injury scores after renal IR. IL-11R deficiency (IL-11R KO) or IL-11 neutralization prevented renal protection with isoflurane postconditioning in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Staining, Neutralization

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. Representative photomicrographs of four to six experiments for immunohistochemistry (brown staining) for neutrophil infiltration (200×) from kidneys IL-11 receptor wild-type (IL-11R WT) mice, IL-11 receptor deficient (IL-11R KO) mice and IL-11R WT mice pretreated with IL-11 neutralizing antibody and subjected to 30 min. renal ischemia and 24-h reperfusion. B. Quantifications of infiltrated neutrophils per 200× field in the kidneys of mice after renal ischemia reperfusion (RIR). * P < 0.05 vs. vehicle-treated pentobarbital anesthetized mice subjected to RIR. Error bars represent 1 SD. IL-11R WT mice anesthetized with pentobarbital after renal ischemia showed heavy neutrophil infiltration. Isoflurane postconditioning significantly attenuated renal tubular neutrophil infiltration after RIR. IL-11 deficiency (IL-11R KO) or IL-11 neutralization attenuated these reductions in renal neutrophil infiltration with isoflurane post-conditioning in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Immunohistochemistry, Staining, Neutralization

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. Representative photomicrographs of four to six experiments for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (representing apoptotic nuclei, magnification 100×) from kidneys IL-11 receptor wild-type (IL-11R WT) mice, IL-11 receptor deficient (IL-11R KO [knockout]) mice and IL-11R WT mice pretreated with IL-11 neutralizing antibody and subjected to 30 min renal ischemia and 24-h reperfusion. B. Quantifications of apoptotic cells per 100× field in the kidneys of mice after renal ischemia reperfusion (RIR). * P < 0.05 vs. vehicle-treated pentobarbital anesthetized mice subjected to RIR. Error bars represent 1 SD. IL-11R WT mice anesthetized with pentobarbital after renal ischemia showed numerous TUNEL positive cells. Isoflurane postconditioning significantly attenuated renal tubular apoptosis after RIR. IL-11 deficiency (IL-11R KO) or IL-11 neutralization attenuated these reductions in renal tubular apoptosis with isoflurane postconditioning in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: TUNEL Assay, Staining, Knock-Out, Neutralization

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: Collectively, our data suggest that isoflurane anesthesia increases interleukin (IL)-11 messenger RNA (mRNA) and protein synthesis via TGF-β1 signaling. We propose that IL-11 synthesized then subsequently activates IL-11R in neighboring renal tubules, or endothelial cells to induce cytoprotective signaling. Since previous studies have shown that IL-11 reduces the activity of a well-known proinflammatory transcription factor NF-kB49;50, it is highly possible that IL-11 generated with isoflurane treatment may also attenuate NF-kB activity to protect against renal inflammation and injury after acute kidney injury. SMADs are intracellular proteins that transduce extracellular signals from TGF-β1 to the nucleus to initiate downstream gene transcription. Hypothetical pathways (e.g., NF-kB inhibition) leading to cytoprotection are shown in dashed lines. We previous showed that IL-11 produces renal protection by direct induction of sphingosine kinase-1 via nuclear translocation of HIF-1α.14

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s Modified Eagle Media/F12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Invitrogen) at 37°C in a 100% humidified atmosphere of 5% carbon dioxide-95% air.

Techniques: Synthesized, Activity Assay, Generated, Inhibition, Translocation Assay

Journal: Stem cell research & therapy

Article Title: TMT-based quantitative proteomic analysis of spheroid cells of endometrial cancer possessing cancer stem cell properties.

doi: 10.1186/s13287-023-03348-x

Figure Lengend Snippet: Fig. 8 Functional assays of selected targets, PFKFB3, GPRC5A and HK2. A–C The mRNA levels of PFKFB3, GPRC5A and HK2 were detected by qRT-PCR. D The expression levels of PFKFB3, GPRC5A and HK2 were detected by Western blot (Full-length blots are presented in supplementary material). E qRT-PCR results for Ishikawa and HEC1A transfected with HK2-siRNA-1, HK2-siRNA-2, HK2-siRNA-3 and NC. F Western blot analysis revealed significant downregulation in HK2-siRNA cells in comparison with the controls (Full-length blots are presented in supplementary material 1 and 2). G CCK-8 assays for Ishikawa and HEC1A cells transfected with HK2-siRNA or NC. H The number of spheroids for Ishikawa and HEC1A transfected with HK2-siRNA or NC in spheroid formation assay. I KEGG biochemical classification for HK2-siRNA endometrial cancer cells (*P < 0.05, **P < 0.01, *** P < 0.001)

Article Snippet: Primary antibodies of western blotting including HK2 (1:1,000, rabbit anti-human, 2A11C3, #66,974–1-Ig; Proteintech Group, INC, USA), GPRC5A (1:1,000, rabbit anti-human, #10,309–1-AP; Proteintech Group, INC, USA), PFKFB3 (1:1,000, rabbit anti-human, #13,763– 1-AP; Proteintech Group, INC, USA), and GAPDH (1:5,000, mouse anti-human, conjugated with HRP, 1E6D9, #60,004–1-Ig; Proteintech Group, INC, USA).

Techniques: Functional Assay, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Comparison, CCK-8 Assay, Tube Formation Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dietary branched-chain amino acid restriction alters fuel selection and reduces triglyceride stores in hearts of Zucker fatty rats

doi: 10.1152/ajpendo.00334.2019

Figure Lengend Snippet: Effect of dietary branched-chain amino acid (BCAA) restriction on cardiac triglyceride (TG) concentrations, gene expression, and protein abundance. A: Zucker lean rats and Zucker fatty rats (ZFRs) were fed a chow diet (lean control and obese control, respectively) or BCAA-restricted diet (lean restr and obese restr, respectively) and cardiac TG concentrations were measured. n = 9–15 per group. *P < 0.05 vs. lean control. B: mRNA levels in perfused hearts from ZFR fed a chow diet (obese control) or BCAA-restricted diet (obese restr). C: representative immunoblots of glucose transporter (GLUT) 1, GLUT4, hexokinase (HK) II, phosphorylated AKT (p-AKT), and AKT with indicated loading controls and corresponding densitometric measurements. n = 7–8 per group. Data represent mean ± SE. *P < 0.05 for diet effect. CPT, carnitine palmitoyltransferase.

Article Snippet: Membranes were blocked with 5% milk for 1 h before probing with primary antibodies for AKT (9272), phosphorylated AKT (p-AKT; 9271), HKII (2867), pan-actin (8456) (Cell Signaling), GLUT1 (ab652), GLUT4 (ab654; Abcam), or β-tubulin (T8328; Sigma).

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: NOX2-Induced High Glycolytic Activity Contributes to the Gain of COL5A1-Mediated Mesenchymal Phenotype in GBM

doi: 10.3390/cancers14030516

Figure Lengend Snippet: NOX2 induces the activation of HK2-dependent glycolysis in human glioma cells. ( a ) The levels of extracellular acidification rate (ECAR) for glycolysis of glucose and ( b ) quantification of ECAR levels in human glioma cells treated with NOX2 siRNA or control siRNA. ( c ) Representative immunoblot images and ( d ) quantification of NOX2, HK2, and LDH-A protein levels in human glioma cells treated with NOX2 siRNA or control siRNA. β-actin was used as a loading control. ( e ) The levels of extracellular acidification rate (ECAR) for glycolysis of glucose and ( f ) quantification of ECAR levels in human glioma cells with NOX2 over-expression or control. ( g ) Representative immunoblot images and ( h ) quantification of NOX2, HK2, and LDH-A protein levels in human glioma cells with NOX2 over-expression or control. Β-actin was used as a loading control. The results are representative of three independent experiments and are shown as the mean ± SEM. ** p < 0.01, * p < 0.05; ANOVA or Student’s two-tailed t -test.

Article Snippet: Sections were permeabilized in 0.5% Triton-X (T8787, Sigma-Aldrich, St. Louis, MO, USA), blocked in CAS-BlockTM Histochemical Reagent (008120, Thermo Fisher Scientific, Waltham, MA, USA) and then stained with the following antibodies: monoclonal rabbit anti-NOX2 (1:100) (ab129068, Abcam, Cambridge, UK) in Figure 1, monoclonal mouse anti-NOX2 (1:100) (NBP1-41012, Novus, Littleton, CO, USA) in Figures 4 and 6, monoclonal rabbit anti-HK2 antibody (1:100) (#2024, Cell Signaling Technology, Danvers, MA, USA), polyclonal rabbit anti-COL5A1 antibody (1:100) (#37304, Cell Signaling Technology), polyclonal rabbit anti-GFAP (1:100) (SAB5700611, Sigma-Aldrich), and monoclonal mouse anti-GFAP (1:100) (#3670, Cell Signaling Technology).

Techniques: Activation Assay, Control, Western Blot, Over Expression, Two Tailed Test

Journal: Cancers

Article Title: NOX2-Induced High Glycolytic Activity Contributes to the Gain of COL5A1-Mediated Mesenchymal Phenotype in GBM

doi: 10.3390/cancers14030516

Figure Lengend Snippet: Inhibition of NOX2 suppresses the activation of HK2-dependent glycolysis in human glioma cells. ( a ) The levels of extracellular acidification rate (ECAR) for glycolysis of glucose and ( b ) quantification of ECAR levels in human glioma cells treated with GSK2795039 or DMSO. ( c ) Representative immunoblot images and ( d ) quantification of NOX2, HK2, and LDH-A protein levels in human glioma cells treated with GSK2795039 or DMSO. β-actin was used as a loading control. The results are representative of three independent experiments and are shown as the mean ± SEM. ** p < 0.01, * p < 0.05; ANOVA or Student’s two-tailed t -test.

Article Snippet: Sections were permeabilized in 0.5% Triton-X (T8787, Sigma-Aldrich, St. Louis, MO, USA), blocked in CAS-BlockTM Histochemical Reagent (008120, Thermo Fisher Scientific, Waltham, MA, USA) and then stained with the following antibodies: monoclonal rabbit anti-NOX2 (1:100) (ab129068, Abcam, Cambridge, UK) in Figure 1, monoclonal mouse anti-NOX2 (1:100) (NBP1-41012, Novus, Littleton, CO, USA) in Figures 4 and 6, monoclonal rabbit anti-HK2 antibody (1:100) (#2024, Cell Signaling Technology, Danvers, MA, USA), polyclonal rabbit anti-COL5A1 antibody (1:100) (#37304, Cell Signaling Technology), polyclonal rabbit anti-GFAP (1:100) (SAB5700611, Sigma-Aldrich), and monoclonal mouse anti-GFAP (1:100) (#3670, Cell Signaling Technology).

Techniques: Inhibition, Activation Assay, Western Blot, Control, Two Tailed Test

Journal: Cancers

Article Title: NOX2-Induced High Glycolytic Activity Contributes to the Gain of COL5A1-Mediated Mesenchymal Phenotype in GBM

doi: 10.3390/cancers14030516

Figure Lengend Snippet: High levels of NOX2 contribute to high levels of glucose uptake and HK2 expression in patients with GBM. ( a , b ) Representative 18 F-FDG PET /MRI images ( a ) and quantification of maximum TBR levels for 18 F-FDG uptake ( b ) in brains from patients with GBM (G4), G3 glioma (G3), G2 glioma (G2), and G1 glioma (G1). ( c ) Representative immunofluorescence images of NOX2 and HK2 staining in patients with GBM (G4), G3 glioma (G3), G2 glioma (G2), and G1 glioma (G1) showing NOX2 (red), HK2 (green), and DAPI-stained nuclei (blue) (n = 3 per group, n = 10 images per individual subject). Scale bars, 20 μm. White arrows indicate NOX2 and HK2-positive cells. ( d , e ) Relative intensity of HK2-positive staining ( d ) and quantification of NOX2 and HK2-positive glioma cells ( e ) from ( c ). ( f ) The levels of HK2 mRNA in patients with GBM and LGG datasets from TCGA. Wilcoxon test with two-sided and one-sided. ( g ) Spearman’s correlation coefficient analysis between NOX2 and HK2 genes in patients (n = 693) with GBM and LGG dataset from TCGA. R = 0.63, p < 0.0001. ( h ) The survival curve of patients with low (black) (n = 347) and high levels (red) (n = 346) of HK2 mRNA (n = 693) from GBM and LGG dataset from TCGA. The two groups were divided according to median value. p < 0.0001; Kaplan–Meier estimation test with two-sided and one-sided. The results are representative of three independent experiments and are shown as the mean ± SEM. *** p < 0.001, ** p < 0.01, * p < 0.05; ANOVA or Student’s two-tailed t -test.

Article Snippet: Sections were permeabilized in 0.5% Triton-X (T8787, Sigma-Aldrich, St. Louis, MO, USA), blocked in CAS-BlockTM Histochemical Reagent (008120, Thermo Fisher Scientific, Waltham, MA, USA) and then stained with the following antibodies: monoclonal rabbit anti-NOX2 (1:100) (ab129068, Abcam, Cambridge, UK) in Figure 1, monoclonal mouse anti-NOX2 (1:100) (NBP1-41012, Novus, Littleton, CO, USA) in Figures 4 and 6, monoclonal rabbit anti-HK2 antibody (1:100) (#2024, Cell Signaling Technology, Danvers, MA, USA), polyclonal rabbit anti-COL5A1 antibody (1:100) (#37304, Cell Signaling Technology), polyclonal rabbit anti-GFAP (1:100) (SAB5700611, Sigma-Aldrich), and monoclonal mouse anti-GFAP (1:100) (#3670, Cell Signaling Technology).

Techniques: Expressing, Immunofluorescence, Staining, Two Tailed Test

Journal: Cancers

Article Title: NOX2-Induced High Glycolytic Activity Contributes to the Gain of COL5A1-Mediated Mesenchymal Phenotype in GBM

doi: 10.3390/cancers14030516

Figure Lengend Snippet: NOX2 and HK2 induce COL5A1-mediated mesenchymal phenotype in human glioma cells. ( a ) The levels of COL5A1 and FN1 mRNA and ( b ) representative immunoblot images (left) and quantification (right) of COL5A1 and FN1 protein levels in human glioma cells treated with NOX2 siRNA or control siRNA. β-actin was used as a loading control. ( c ) The levels of COL5A1 and FN1 mRNA and ( d ) representative immunoblot images (left) and quantification (right) of COL5A1 and FN1 protein levels in human glioma cells with NOX2 over-expression (NOX2) or control (Control). β-actin was used as a loading control. ( e ) Representative images of cellular morphology in U87MG glioma cells with NOX2 over-expression (HK2) or control (Control) (left). Quantification of spindle shape length in U87MG glioma cells with NOX2 over-expression or control (right). Scale bars, 20 μm. ( f ) The levels of COL5A1 and FN1 mRNA and ( g ) representative immunoblot images (left) and quantification (right) of COL5A1 and FN1 protein levels in U87MG glioma cells with HK2 over-expression (HK2) or control (Control). ( h ) Representative images of cellular morphology in U87MG glioma cells with HK2 over-expression (HK2) or control (Control) (left). Scale bars, 20 μm. Quantification of spindle shape length in human glioma cells with HK2 over-expression (HK2) or control (Control) (right). The results are representative of three independent experiments and are shown as the mean ± SEM. * p < 0.05; Student’s two-tailed t -test.

Article Snippet: Sections were permeabilized in 0.5% Triton-X (T8787, Sigma-Aldrich, St. Louis, MO, USA), blocked in CAS-BlockTM Histochemical Reagent (008120, Thermo Fisher Scientific, Waltham, MA, USA) and then stained with the following antibodies: monoclonal rabbit anti-NOX2 (1:100) (ab129068, Abcam, Cambridge, UK) in Figure 1, monoclonal mouse anti-NOX2 (1:100) (NBP1-41012, Novus, Littleton, CO, USA) in Figures 4 and 6, monoclonal rabbit anti-HK2 antibody (1:100) (#2024, Cell Signaling Technology, Danvers, MA, USA), polyclonal rabbit anti-COL5A1 antibody (1:100) (#37304, Cell Signaling Technology), polyclonal rabbit anti-GFAP (1:100) (SAB5700611, Sigma-Aldrich), and monoclonal mouse anti-GFAP (1:100) (#3670, Cell Signaling Technology).

Techniques: Western Blot, Control, Over Expression, Two Tailed Test

Journal: Cancers

Article Title: NOX2-Induced High Glycolytic Activity Contributes to the Gain of COL5A1-Mediated Mesenchymal Phenotype in GBM

doi: 10.3390/cancers14030516

Figure Lengend Snippet: NOX2 and HK2 contributes to the gain of the COL5A1-mediated mesenchymal phenotype in patients with GBM. ( a ) Representative immunofluorescence images of NOX2 and COL5A1 staining in patients with GBM (G4), G3 glioma (G3), G2 glioma (G2), and G1 glioma (G1) showing NOX2 (red), COL5A1 (green), and DAPI-stained nuclei (blue) (n = 3 per group, n = 10 images per individual subject). Scale bars, 20 μm. White arrows indicate NOX2 and COL5A1-positive cells. ( b ) Representative immunofluorescence images of HK2 and COL5A1 staining in patients with GBM (G4), G3 glioma (G3), G2 glioma (G2), and G1 glioma (G1) showing COL5A1 (red), HK2 (green), and DAPI-stained nuclei (blue) (n = 3 per group, n = 10 images per individual subject). Scale bars, 20 μm. White arrows indicate HK2 and COL5A1-positive cells. ( c , d ) Relative intensity of COL5A1-positive staining ( c ) and quantification of NOX2 and COL5A1-positive glioma cells ( d ) from ( a ). ( e ) Quantification of HK2 and COL5A1-positive glioma cells from ( b ). ( f ) A Spearman’s correlation coefficient analysis between HK2 and COL5A1 gene patients (n = 693) with GBM and LGG datasets from TCGA. R = 0.28, p < 0.0001. ( g ) Spearman’s correlation coefficient analysis between HK2 and COL5A1 gene patients (n = 693) with GBM and LGG datasets from TCGA. R = 0.38, p < 0.0001. The results are representative of three independent experiments and are shown as the mean ± SEM. ** p < 0.01, * p < 0.05; ANOVA or Student’s two-tailed t -test.

Article Snippet: Sections were permeabilized in 0.5% Triton-X (T8787, Sigma-Aldrich, St. Louis, MO, USA), blocked in CAS-BlockTM Histochemical Reagent (008120, Thermo Fisher Scientific, Waltham, MA, USA) and then stained with the following antibodies: monoclonal rabbit anti-NOX2 (1:100) (ab129068, Abcam, Cambridge, UK) in Figure 1, monoclonal mouse anti-NOX2 (1:100) (NBP1-41012, Novus, Littleton, CO, USA) in Figures 4 and 6, monoclonal rabbit anti-HK2 antibody (1:100) (#2024, Cell Signaling Technology, Danvers, MA, USA), polyclonal rabbit anti-COL5A1 antibody (1:100) (#37304, Cell Signaling Technology), polyclonal rabbit anti-GFAP (1:100) (SAB5700611, Sigma-Aldrich), and monoclonal mouse anti-GFAP (1:100) (#3670, Cell Signaling Technology).

Techniques: Immunofluorescence, Staining, Two Tailed Test