Journal: JCI Insight

Article Title: KLF11 protects against abdominal aortic aneurysm through inhibition of endothelial cell dysfunction

doi: 10.1172/jci.insight.141673

Figure Lengend Snippet: ( A and B ) The production of superoxide was determined in HAECs. HAECs were infected with Ad- lacZ or Ad- KLF11 , or Ad-sh lacZ or Ad-sh KLF11 (10 MOI). After 48 hours, they were treated with TNF-α (2 ng/mL) or AngII (1 μM) for 2 hours in the presence of superoxide detection solution (fluorescent probes). ( C – E ) HAECs were infected with Ad- GFP or Ad- KLF11 (10 MOI) or transfected with control siRNA (siControl) or KLF11 siRNA (si KLF11 , 20 μM). After 48 hours, they were stimulated with TNF-α (2 ng/mL) or AngII (1 μM) for 24 hours. ( C and D ) The mRNA levels of NOX1 , NOX2 , NOX4 , and NOX5 were determined by qPCR. ( E ) Western blot to determine the expression of NOX2 in HAECs. ( F ) HAECs were transfected with siControl, si KLF11 or si KLF11 + NOX2 siRNA (si NOX2 ) (20 μM). After 48 hours, they were treated with TNF-α (2 ng/mL) or AngII (1 μM) for 2 hours in the presence of superoxide detection solution. ( G and H ) Representative DHE staining and quantification of superoxide production in HAECs. HAECs were transfected with siControl, si KLF11 , or si KLF11 +si NOX2 . After 48 hours, they were pretreated with NOX2 inhibitor (NOX2i, GSK2795039, 1 μM) for 1 hour, then stimulated with TNF-α (2 ng/mL) or AngII (1 μM) for 2 hours, followed by DHE staining of superoxide (red) and immunofluorescence staining of VE-cadherin (green). Nuclei stained by DAPI are shown in blue. Scale bar: 20 μm. ( I ) HAECs were infected with Ad- lacZ or Ad-flag- KLF11 . After 48 hours, they were stimulated with TNF-α (2 ng/mL) for 4 hours, followed by ChIP assay using an antibody against flag or IgG. Data are presented as mean ± SEM. Two-way ANOVA followed by Holm-Sidak post hoc analysis ( A – D , F , H , and I ).

Article Snippet: NOX2 antibody (NBP2-67680) was purchased from Novus Biologicals.

Techniques: Infection, Transfection, Control, Western Blot, Expressing, Staining, Immunofluorescence

Journal: JCI Insight

Article Title: KLF11 protects against abdominal aortic aneurysm through inhibition of endothelial cell dysfunction

doi: 10.1172/jci.insight.141673

Figure Lengend Snippet: ( A – E ) Human aortic smooth muscle cells (HASMCs) were treated for 24 hours with the conditioned media from ECs (EC-CM) that had been transfected with siControl, si KLF11 , or si KLF11 +si NOX2 (20 μM), or infected with Ad- GFP or Ad- KLF11 (10 MOI) and subsequently stimulated for 1 hour with TNF-α (2 ng/mL) 48 hours after siRNA transfection or adenovirus infection and cultured in fresh opti-MEM for an additional 4 hours. ( A – D ) qPCR ( A and C ) and Western blot ( B and D ) to examine expression of SMC-specific contractile markers (smooth muscle α-actin [SMA], calponin, and smooth muscle 22–α [SM22α]), proinflammatory cytokines (MCP-1 and IL-6), and metalloproteinases (MMP2 and MMP9). ( E ) HASMCs were cultured in EC-CM for 48 hours, followed by immunostaining of TUNEL. Scale bar: 20 μm. ( F – H ) Schematics of the in vitro coculture system using a Transwell. HAECs (upper chamber) transfected with siControl, si KLF11 (20 μM), or si KLF11 +si NOX2 or infected with Ad- GFP or Ad- KLF11 (10 MOI) were cultured for 48 hours followed by TNF-α (2 ng/mL) stimulation for 1 hour separately from HASMCs, changed to fresh opti-MEM, and then cocultured with HASMCs (bottom) in fresh opti-MEM for 24 hours. The expression of BAX in HASMCs was assayed by Western blot ( G and H ). Data are mean ± SEM from 3 independent experiments. Two-way ANOVA followed by Holm-Sidak post hoc analysis ( A , C , E , G , and H ).

Article Snippet: NOX2 antibody (NBP2-67680) was purchased from Novus Biologicals.

Techniques: Transfection, Infection, Cell Culture, Western Blot, Expressing, Immunostaining, TUNEL Assay, In Vitro

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: The levels of NOX2 are elevated in patients with GBM compared to low-grade glioma. ( a ) Representative immunofluorescence images of NOX2 staining in patients with GBM (G4), G3 glioma (G3), G2 glioma (G2), and G1 glioma (G1) showing NOX2 (green), GFAP (red, glioma cells), and DAPI-stained nuclei (blue) (n = 3 per group, n = 10 images per individual subject). Scale bars, 20 μm. White arrows indicate NOX2 and GFAP-positive glioma cells. ( b , c ) Relative intensity of NOX2-positive staining ( b ) and quantification of NOX2 and GFAP-positive glioma cells ( c ) from ( a ). ( d ) The levels of NOX2 mRNA in patients with GBM and LGG dataset from TCGA. Wilcoxon test with two-sided and one-sided. ( e ) The survival curve of patients with low (black) (n = 347) and high levels (red) (n = 346) of NOX2 mRNA (n = 693) from GBM and LGG datasets 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: For overexpression of human NOX2, U87MG cells were seeded (2 × 10 5 cells/6-well cell culture plates) and transfected with pCMV6-AC-GFP constructs containing human NOX2 (NM_000397) (RG207544, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: 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 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: For overexpression of human NOX2, U87MG cells were seeded (2 × 10 5 cells/6-well cell culture plates) and transfected with pCMV6-AC-GFP constructs containing human NOX2 (NM_000397) (RG207544, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

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: For overexpression of human NOX2, U87MG cells were seeded (2 × 10 5 cells/6-well cell culture plates) and transfected with pCMV6-AC-GFP constructs containing human NOX2 (NM_000397) (RG207544, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

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: For overexpression of human NOX2, U87MG cells were seeded (2 × 10 5 cells/6-well cell culture plates) and transfected with pCMV6-AC-GFP constructs containing human NOX2 (NM_000397) (RG207544, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

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: For overexpression of human NOX2, U87MG cells were seeded (2 × 10 5 cells/6-well cell culture plates) and transfected with pCMV6-AC-GFP constructs containing human NOX2 (NM_000397) (RG207544, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

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: For overexpression of human NOX2, U87MG cells were seeded (2 × 10 5 cells/6-well cell culture plates) and transfected with pCMV6-AC-GFP constructs containing human NOX2 (NM_000397) (RG207544, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Two Tailed Test