Journal: International Journal of Medical Sciences

Article Title: Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation

doi: 10.7150/ijms.97164

Figure Lengend Snippet: Summary of the genetic findings of our 8 patients

Article Snippet: The experimental groups included the control group (no drug treatment), LPS group (cells stimulated with 5 μg/mL LPS for 48 hours to induce neuroinflammation), LPS+NC group (cells stimulated with LPS and transfected with an empty adenoviral vector), LPS+ Col4a2 Wt group (cells stimulated with LPS and transfected with the Col4a2 Wt -AdV vector), LPS+Col4a2 Mut group (cells stimulated with LPS and transfected with the Col4a2 Mut -AdV vector carrying the c.1838G>T, p.G613V mutation), and LPS+ Col4a2 Mut +WP1066 group (cells treated with the JAK2/STAT inhibitor WP1066 (5 μM, HY-15312; MCE, China) after stimulation with LPS and transfection with the Col4a2 Mut -AdV vector).

Techniques:

Journal: International Journal of Medical Sciences

Article Title: Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation

doi: 10.7150/ijms.97164

Figure Lengend Snippet: Mutation of Col4a2 promotes inflammation and astrocyte activation in CTX-TNA cells. CTX-TNA cells were stimulated with LPS and transfected with the Col4a2 Wt -AdV vector or the Col4a2 Mut -AdV vector. (A) The fluorescence intensity of GFAP (red) was measured in 5 different groups, with 50 CTX-TNA cells per group from 3 repeated experiments. The scale bar represents 25 μm. (B) Quantitative graphs displaying the mean fluorescence intensity of the GFAP-positive cells in (A). (C-E) Enzyme-linked immunosorbent assays were used to quantify IL-1β, IL-6, and TNF-α levels in the supernatant of CTX-TNA cells from 3 repeated experiments. The figure includes results from one-way ANOVA with calculated p values. (F) Western blot analysis was performed for GFAP, IL-1β, IL-6, TNF-α, JAK2, p-JAK2, STAT3, and p-STAT3. The proteins for the western blot analysis were extracted from cell lysates. **: vs. con, p<0.01; ++: vs. LPS+NC, p<0.01; ##: vs. LPS+ Col4a2 Wt , p <0.01.

Article Snippet: The experimental groups included the control group (no drug treatment), LPS group (cells stimulated with 5 μg/mL LPS for 48 hours to induce neuroinflammation), LPS+NC group (cells stimulated with LPS and transfected with an empty adenoviral vector), LPS+ Col4a2 Wt group (cells stimulated with LPS and transfected with the Col4a2 Wt -AdV vector), LPS+Col4a2 Mut group (cells stimulated with LPS and transfected with the Col4a2 Mut -AdV vector carrying the c.1838G>T, p.G613V mutation), and LPS+ Col4a2 Mut +WP1066 group (cells treated with the JAK2/STAT inhibitor WP1066 (5 μM, HY-15312; MCE, China) after stimulation with LPS and transfection with the Col4a2 Mut -AdV vector).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation

doi: 10.7150/ijms.97164

Figure Lengend Snippet: Col4a2 mutation promotes inflammation and astrocyte activation in primary astrocytes. Primary astrocytes were stimulated with LPS and transfected with either the Col4a2 Wt -AdV vector or the Col4a2 Mut -AdV vector. (A) The fluorescence intensity of GFAP (red) was measured in 5 different primary astrocyte groups, with 50 cells per group from 3 repeated experiments. The scale bar indicates 25 μm. (B) Quantitative graphs displaying the mean fluorescence intensity of the GFAP-positive cells in (A). (C-E) Enzyme-linked immunosorbent assays were used to measure IL-1β, IL-6, and TNF-α levels in primary astrocyte supernatants from 3 repeated experiments. The figure includes one-way ANOVA results with corresponding p values. (F) Western blot analysis was performed for GFAP, IL-1β, IL-6, TNF-α, JAK2, p-JAK2, STAT3, and p-STAT3. The proteins for the western blot analysis were extracted from cell lysates. **: vs. control, p<0.01; ++: vs. LPS+NC, p<0.01; ##: vs. LPS+ Col4a2 Wt , p <0.01.

Article Snippet: The experimental groups included the control group (no drug treatment), LPS group (cells stimulated with 5 μg/mL LPS for 48 hours to induce neuroinflammation), LPS+NC group (cells stimulated with LPS and transfected with an empty adenoviral vector), LPS+ Col4a2 Wt group (cells stimulated with LPS and transfected with the Col4a2 Wt -AdV vector), LPS+Col4a2 Mut group (cells stimulated with LPS and transfected with the Col4a2 Mut -AdV vector carrying the c.1838G>T, p.G613V mutation), and LPS+ Col4a2 Mut +WP1066 group (cells treated with the JAK2/STAT inhibitor WP1066 (5 μM, HY-15312; MCE, China) after stimulation with LPS and transfection with the Col4a2 Mut -AdV vector).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Western Blot, Control

Journal: International Journal of Medical Sciences

Article Title: Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation

doi: 10.7150/ijms.97164

Figure Lengend Snippet: Col4a2 mutation promotes inflammation and astrocyte activation by stimulating JAK/STAT signaling in CTX-TNA cells. CTX-TNA cells were stimulated with LPS and transfected with the Col4a2 Wt -AdV vector or the Col4a2 Mut -AdV vector or treated with WP1066 (a JAK2/STAT inhibitor). (A) The fluorescence intensity of GFAP was observed in 6 different groups (50 CTX-TNA cells per group) from 3 repeated experiments. The scale bar represents 25 μm. (B) Quantitative graphs displaying the mean fluorescence intensity of the GFAP-positive cells in (A). The figure includes the results of one-way ANOVA with calculated p values. (C) Western blot analysis of GFAP, IL-1β, IL-6, TNF-α, JAK2, p-JAK2, STAT3, and p-STAT3 was performed. The proteins for the western blot analysis were extracted from cell lysates. **: vs. con, p<0.01; ++: vs. LPS+NC, p<0.01; ##: vs. LPS+ Col4a2 Wt , p <0.01; ^^: vs. LPS+ Col4a2 Mut , p<0.01.

Article Snippet: The experimental groups included the control group (no drug treatment), LPS group (cells stimulated with 5 μg/mL LPS for 48 hours to induce neuroinflammation), LPS+NC group (cells stimulated with LPS and transfected with an empty adenoviral vector), LPS+ Col4a2 Wt group (cells stimulated with LPS and transfected with the Col4a2 Wt -AdV vector), LPS+Col4a2 Mut group (cells stimulated with LPS and transfected with the Col4a2 Mut -AdV vector carrying the c.1838G>T, p.G613V mutation), and LPS+ Col4a2 Mut +WP1066 group (cells treated with the JAK2/STAT inhibitor WP1066 (5 μM, HY-15312; MCE, China) after stimulation with LPS and transfection with the Col4a2 Mut -AdV vector).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation

doi: 10.7150/ijms.97164

Figure Lengend Snippet: Col4a2 mutation promotes inflammation and astrocyte activation by stimulating JAK/STAT signaling in primary astrocytes. Primary astrocytes were stimulated with LPS and transfected with the Col4a2 Wt -AdV vector or the Col4a2 Mut -AdV vector or treated with WP1066 (a JAK2/STAT inhibitor). (A) The fluorescence intensity of GFAP in six different primary astrocyte groups (50 cells per group) from 3 repeated experiments is shown. The scale bar represents 25 μm. (B) Quantitative graphs displaying the mean fluorescence intensity of the GFAP-positive cells in (A). One-way ANOVA with calculated p values is presented in the figure. (C) Western blot analysis was conducted for GFAP, IL-1β, IL-6, TNF-α, JAK2, p-JAK2, STAT3, and p-STAT3. The proteins for the western blot analysis were extracted from cell lysates. **: vs. control, p<0.01; ++: vs. LPS+NC, p<0.01; ##: vs. LPS+ Col4a2 Wt , p <0.01; ^^: vs. LPS+ Col4a2 Mut , p<0.01.

Article Snippet: The experimental groups included the control group (no drug treatment), LPS group (cells stimulated with 5 μg/mL LPS for 48 hours to induce neuroinflammation), LPS+NC group (cells stimulated with LPS and transfected with an empty adenoviral vector), LPS+ Col4a2 Wt group (cells stimulated with LPS and transfected with the Col4a2 Wt -AdV vector), LPS+Col4a2 Mut group (cells stimulated with LPS and transfected with the Col4a2 Mut -AdV vector carrying the c.1838G>T, p.G613V mutation), and LPS+ Col4a2 Mut +WP1066 group (cells treated with the JAK2/STAT inhibitor WP1066 (5 μM, HY-15312; MCE, China) after stimulation with LPS and transfection with the Col4a2 Mut -AdV vector).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Western Blot, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TGF-β-induced regulatory T cells directly suppress B cell responses through a non-cytotoxic mechanism

doi: 10.4049/jimmunol.1501740

Figure Lengend Snippet: Both iTreg and nTreg directly suppress B cells. B cells were isolated from C57BL/6, stimulated with (Baseline) or without LPS (2 µg/ml) (B only) in the presence or absence of nTreg, iTreg and CD4med (ratio of T to B cells was 1:2 to 1:4). (A) The expression of B cell activation (CD69, CD80, and CD86) and differentiation (CD138) markers was detected after 20 and 48 hours of culture by flow cytometry. Typical FACS plots (left) and summary data (right) were shown. (B) Fresh B cells labeled with carboxyfluorescein succinimidyl ester (CFSE) were cultured with nTreg, iTreg or CD4med cells (The ratio of T cells: B cell was 1:2 to 1:8) in the presence of LPS (2µg/mL), B-cell proliferation was determined by the CFSE dilution rates after 2 days of culture. In lower panel, CFSE-labeled B cells were pre-incubated with LPS (2µg/mL) for 24 hours and then cells were extensively washed out to remove LPS. Treg or CD4med were co-cultured with LPS pretreat-B cells for another 2 days, the proliferation was analyzed by the flow cytometry. (C and D) The supernatants were collected from above cultural systems after 3 days culture, and the IgG, IgM secretion was detected by ELISA. The data indicate the Mean ± SEM of 3 separated experiments (*p<0.05, **p<0.01, ***p<0.001; baseline or CD4med vs. Treg group).

Article Snippet: Then B cells were stimulated with or without Lipopolysaccharides (LPS) (2µg/mL, Escherichia coli serotype 0111: B4, Sigma-Aldrich) in the presence or absence of nTreg, iTreg or CD4 med cells.

Techniques: Isolation, Expressing, Activation Assay, Flow Cytometry, Labeling, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TGF-β-induced regulatory T cells directly suppress B cell responses through a non-cytotoxic mechanism

doi: 10.4049/jimmunol.1501740

Figure Lengend Snippet: Inhibitor role of iTreg on B cells proliferation is mostly dependent upon TGF-β signal in vivo, and partially on IL-10 signal. (A–D) NZM2328 fresh B cells were stimulated with LPS (2µg/mL) for 24 hours, then co-transferred with nTreg, iTreg or CD4med (2:1 ratio) into Rag1−/− mice, in some groups, ALK5i (1mg/kg), anti-IL-10R antibody (1mg/kg), DMSO (for Alk5i control) or control IgG were i.p. injected at day 0 and day 2. The mice were sacrificed and spleen B cells were harvested and prepared to test Ki-67 expression on B cells by flow cytometry at days 3. There were five mice in each group. The data indicated Mean ± SEM of two separate experiments. *p<0.05, **p<0.01 means control cell group vs. iTreg group after cell transfer.

Article Snippet: Then B cells were stimulated with or without Lipopolysaccharides (LPS) (2µg/mL, Escherichia coli serotype 0111: B4, Sigma-Aldrich) in the presence or absence of nTreg, iTreg or CD4 med cells.

Techniques: In Vivo, Injection, Expressing, Flow Cytometry

Journal: Immunology

Article Title: Expression and regulation of interleukin-33 in human monocytes

doi: 10.1111/j.1365-2567.2009.03221.x

Figure Lengend Snippet: Interleukin-33 (IL-33) protein is closely associated with the nucleus in THP-1 monocytes undergoing apoptosis. Immunocytochemistry was performed on unstimulated THP-1 monocytes (control) and on cells stimulated with 100 ng/ml Escherichia coli (Ec) or Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) for 9 hr followed by exposure to UVB-irradiation to induce apoptosis. The IL-33 was located using an IL-33 antibody and secondary antibody conjugated to fluorescein isothiocyanate (FITC; green). The nuclei were stained with 4′-6′,diamidino-2-phenylindole (DAPI; blue). The upper panels (a) show IL-33 expression alone (green FITC) and the lower panels (b) show IL-33 (green FITC) and nuclei (blue DAPI) in combination. Arrows indicate examples of apoptotic cells. The results are representative of three independent stimulation experiments and have been verified by an independent observer. Scale as depicted on the images.

Article Snippet: Cells were stimulated with 100 ng/ml E. coli LPS (from strain 0111:B4; Invivogen, Calne, UK), 100 ng/ml P. gingivalis (from strain W50, a gift from M. Rangarajan, Queen Mary’s School of Medicine and Dentistry, London, UK), 100 ng/ml TNF-α (R&D Systems, Abingdon, UK), 100 pg/ml IL-1β (R&D Systems) or ATP (Sigma) for between 0·5 and 48 hr.

Techniques: Immunocytochemistry, Irradiation, Staining, Expressing

Journal: Immunology

Article Title: Expression and regulation of interleukin-33 in human monocytes

doi: 10.1111/j.1365-2567.2009.03221.x

Figure Lengend Snippet: Interleukin-33 (IL-33) messenger RNA (mRNA) expression is up-regulated by monocytes in response to lipopolysaccharide (LPS) but not tumour necrosis factor-α (TNF-α) and IL-1β. Quantitative real-time polymerase chain reaction analysis was used to determine IL-33 expression by THP-1 monocytes (a) and primary human monocytes (b) in response to stimulation with 100 ng/ml Escherichia coli or Porphyromonas gingivalis LPS. IL-33 mRNA expression by THP-1 monocytes stimulated with 100 ng/ml TNF-α and 100 pg/ml IL-1β was also measured (c). Levels of mRNA were normalized against RNA polymerase II mRNA levels and fold changes (‘IL-33 expression’) were calculated relative to unstimulated cells at each individual time-point using the ΔΔCt method.20 The data represent the mean values (± SD) from three independent experiments. Significant differences we determined using Student’s t-test on the ΔCt values.20*P < 0·05 relative to unstimulated cells.

Article Snippet: Cells were stimulated with 100 ng/ml E. coli LPS (from strain 0111:B4; Invivogen, Calne, UK), 100 ng/ml P. gingivalis (from strain W50, a gift from M. Rangarajan, Queen Mary’s School of Medicine and Dentistry, London, UK), 100 ng/ml TNF-α (R&D Systems, Abingdon, UK), 100 pg/ml IL-1β (R&D Systems) or ATP (Sigma) for between 0·5 and 48 hr.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Immunology

Article Title: Expression and regulation of interleukin-33 in human monocytes

doi: 10.1111/j.1365-2567.2009.03221.x

Figure Lengend Snippet: Interleukin-33 (IL-33) is not secreted by viable THP-1 monocytes but is released from necrotic cells. THP-1 monocytes were either stimulated with 100 ng/ml Escherichia coli or Porphyromonas gingivalis lipopolysaccharide (LPS) for 9 hr (live control), or stimulated with 100 ng/ml E. coli or P. gingivalis LPS for 3–48 hr followed by induction of necrosis by five rounds of freezing (− 70°) and thawing (38°). Stimulation of the cells in these experiments was confirmed by detection of tumour necrosis factor-α in the isolated supernatants using enzyme-linked immunosorbent assay (not shown). ND = not detected. The data represent the mean values (± SD) from three independent experiments.

Article Snippet: Cells were stimulated with 100 ng/ml E. coli LPS (from strain 0111:B4; Invivogen, Calne, UK), 100 ng/ml P. gingivalis (from strain W50, a gift from M. Rangarajan, Queen Mary’s School of Medicine and Dentistry, London, UK), 100 ng/ml TNF-α (R&D Systems, Abingdon, UK), 100 pg/ml IL-1β (R&D Systems) or ATP (Sigma) for between 0·5 and 48 hr.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Expression and regulation of interleukin-33 in human monocytes

doi: 10.1111/j.1365-2567.2009.03221.x

Figure Lengend Snippet: Effect of adenosine triphosphate (ATP) on interleukin-33 (IL-33) and IL-18 secretion by THP-1 monocytes

Article Snippet: Cells were stimulated with 100 ng/ml E. coli LPS (from strain 0111:B4; Invivogen, Calne, UK), 100 ng/ml P. gingivalis (from strain W50, a gift from M. Rangarajan, Queen Mary’s School of Medicine and Dentistry, London, UK), 100 ng/ml TNF-α (R&D Systems, Abingdon, UK), 100 pg/ml IL-1β (R&D Systems) or ATP (Sigma) for between 0·5 and 48 hr.

Techniques:

Journal: Immunology

Article Title: Expression and regulation of interleukin-33 in human monocytes

doi: 10.1111/j.1365-2567.2009.03221.x

Figure Lengend Snippet: Interleukin-33 (IL-33) protein is expressed in the cytoplasm of lipopolysaccharide (LPS) -stimulated THP-1 monocytes (a) and primary monocytes (b). Immunocytochemistry was performed on monocytes (control) and cells stimulated with 100 ng/ml Escherichia coli (Ec) or Porphyromonas gingivalis (Pg) LPS for 9 hr. The IL-33 was located using an IL-33 antibody and secondary antibody conjugated to fluorescein isothiocyanate (FITC; green) or tetramethyl rhodamine iso-thiocyanate (TRITC; red) and the nuclei were stained with 4′-6′,diamidino-2-phenylindole (DAPI; blue). A series of Z-stack images were taken to confirm the cellular location of IL-33 and the image depicted is representative of findings throughout the Z stack. The upper panels (a) show IL-33 expression alone (green FITC) and the lower panels (b) show IL-33 (red TRITC) and nuclei (blue DAPI) in combination. The results are representative of three independent stimulation experiments and have been verified by an independent observer. Scale as depicted on images.

Article Snippet: Cells were stimulated with 100 ng/ml E. coli LPS (from strain 0111:B4; Invivogen, Calne, UK), 100 ng/ml P. gingivalis (from strain W50, a gift from M. Rangarajan, Queen Mary’s School of Medicine and Dentistry, London, UK), 100 ng/ml TNF-α (R&D Systems, Abingdon, UK), 100 pg/ml IL-1β (R&D Systems) or ATP (Sigma) for between 0·5 and 48 hr.

Techniques: Immunocytochemistry, Staining, Expressing

Journal: Redox Biology

Article Title: Methylglyoxal suppresses microglia inflammatory response through NRF2-IκBζ pathway

doi: 10.1016/j.redox.2023.102843

Figure Lengend Snippet: MGOs are accumulated in activated immune cells. (a) BV2 cells were stimulated with LPS (1 μg/mL) for 24 h. Intracellular MGO level was assessed using a fluorescent probe and detected through flow cytometry. (b) Immunofluorescent stain for intracellular MGO-modified proteins (MGO-p) in BV2 cells stimulated with LPS (1 μg/mL) for 24 h. (c) Mouse splenic CD3 + T cells were isolated and stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibodies for the indicated time. Intracellular MGO levels were determined using a fluorescent probe and analyzed by flow cytometry. (d) Mouse splenic CD3 + T cells were isolated and stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibodies for the indicated time. Intracellular MGO-p were measured using flow cytometry. (e) Flow cytometry analysis of intracellular MGO-p in Th1 (CD4+IFN-γ+), non-Th1(CD4+IFN-γ-), Th17 (CD4+IL-17+), non-Th17 (CD4+IL-17-), Treg (CD4+Foxp3+), and non-Treg (CD4+Foxp3-) cells. * p < 0.05; ** p < 0.01; *** p < 0.001. Ns, not significant. Three independent experiments were performed.

Article Snippet: Cells were stimulated with 1 μg/mL LPS ( E. coli , Sigma Aldrich) or 10 ng/mL IL-4 (214–14, Peprotech) and 10 ng/mL IL-13 (210–13, Peprotech) in the presence of MGO or not for indicated time.

Techniques: Flow Cytometry, Staining, Modification, Isolation

Journal: Redox Biology

Article Title: Methylglyoxal suppresses microglia inflammatory response through NRF2-IκBζ pathway

doi: 10.1016/j.redox.2023.102843

Figure Lengend Snippet: MGO modulates microglia polarization in vitro . (a) Flow cytometry analysis of iNOS expression in BV2 cells treated with LPS and different concentrations of MGO for 24 h. (b) BV2 cells were stimulated with IL-4 plus IL-13 in the presence or absence of MGO for 24 h, and the expression of Arg-1 was analyzed by flow cytometry. (c and d) Flow cytometry analysis of IL-1β and IL-6 expressions in BV2 cells treated with LPS ± 200 μM MGO for 24 h. (e) Real-time PCR analysis of IL-1β and IL-6 expressions in BV2 cells treated with LPS ± 200 μM MGO for 24 h. (f) Flow cytometry and real-time PCR analysis of TNF-α expression in BV2 cells treated with LPS ± 200 μM MGO for 24 h. (g) BV2 cells treated with LPS and 200 μM MGO for 12 or 24 h, multiplex cytokine profiling of supernatants was analyzed by cytometric bead-based immunoassay. * p < 0.05; ** p < 0.01; *** p < 0.001. Ns, not significant. Three independent experiments were performed.

Article Snippet: Cells were stimulated with 1 μg/mL LPS ( E. coli , Sigma Aldrich) or 10 ng/mL IL-4 (214–14, Peprotech) and 10 ng/mL IL-13 (210–13, Peprotech) in the presence of MGO or not for indicated time.

Techniques: In Vitro, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Multiplex Assay, Bead-based Assay

Journal: Redox Biology

Article Title: Methylglyoxal suppresses microglia inflammatory response through NRF2-IκBζ pathway

doi: 10.1016/j.redox.2023.102843

Figure Lengend Snippet: MGO inhibits the capacity of microglia cells to recruit and activate lymphocytes. BV2 cells were stimulated with 1 μg/mL LPS ±200 μM MGO for 12 h. (a and b) The expressions of CD80, CD86, and MHC II on the surface of BV2 cells were measured using flow cytometry. (c and d) BV2 cells were washed three times with PBS and co-cultured with purified mouse splenic T lymphocytes at a ratio of 1:10. After 3 d, the cells were harvested, stained with antibodies against CD4, CD25, and ICOS, and analyzed using flow cytometry. (e) Expressions of CXCL2, CCL7, and CCL12 were detected using real-time PCR. (f) A transwell assay was applied to lymphocytes co-cultured with LPS or LPS plus MGO-stimulated BV2 cells, and cell numbers were counted by flow cytometry. * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001. Ns, not significant. Three independent experiments were performed.

Article Snippet: Cells were stimulated with 1 μg/mL LPS ( E. coli , Sigma Aldrich) or 10 ng/mL IL-4 (214–14, Peprotech) and 10 ng/mL IL-13 (210–13, Peprotech) in the presence of MGO or not for indicated time.

Techniques: Flow Cytometry, Cell Culture, Purification, Staining, Real-time Polymerase Chain Reaction, Transwell Assay

Journal: Redox Biology

Article Title: Methylglyoxal suppresses microglia inflammatory response through NRF2-IκBζ pathway

doi: 10.1016/j.redox.2023.102843

Figure Lengend Snippet: MGO suppresses microglia cell glycolysis by decreasing GLUT1 expression. (a) The absorption spectrum of the complete culture medium with different concentrations of hydrochloric acid (left) and correlation analysis of ratios of absorbance at 415–560 nm with the concentrations of hydrochloric acid (right). (b) Acid concentration in the supernatant of BV2 cells treated with 1 μg/mL LPS ± 200 μM MGO for 24 h. (c) Glucose consumption of BV2 cells treated with 1 μg/mL LPS ± 200 μM MGO was measured after 6- and 24-h incubation. (d) Immunofluorescent stain of GLUT1 on BV2 cells after treatment with LPS ± 200 μM MGO for 24 h. (e) Flow cytometry analysis of GLUT1 on the surface of BV2 cells treated with LPS ± 200 μM MGO for 24 h. (f) BV2 cells were stimulated with 1 μg/mL LPS ± 200 μM MGO for 24 h. Mitochondrial membrane potentials were addressed using JC-1 fluorescent probe and analyzed by flow cytometry. Three independent experiments were performed. * p < 0.05; ** p < 0.01; *** p < 0.001. Ns, not significant.

Article Snippet: Cells were stimulated with 1 μg/mL LPS ( E. coli , Sigma Aldrich) or 10 ng/mL IL-4 (214–14, Peprotech) and 10 ng/mL IL-13 (210–13, Peprotech) in the presence of MGO or not for indicated time.

Techniques: Expressing, Concentration Assay, Incubation, Staining, Flow Cytometry

Journal: Redox Biology

Article Title: Methylglyoxal suppresses microglia inflammatory response through NRF2-IκBζ pathway

doi: 10.1016/j.redox.2023.102843

Figure Lengend Snippet: MGO restrains pro-inflammatory cytokine production by suppressing IκBζ expression. BV2 cells were stimulated with 1 μg/mL LPS and 200 μM MGO for 24 h. Gene expression was determined using RNA sequencing. (a) Heatmap depicting differentially expressed genes between LPS-stimulated and LPS plus MGO-conditioned BV2 cells. (b) GO analysis of differentially expressed genes between the two groups. (c) Heatmap depicting differentially expressed genes related to inflammatory responses. (d and e) GSEA of enriched Toll-like receptor and NF-κB signaling pathways. (f) Real-time PCR and western blot analysis of IκBζ ( Nfkbiz ) expression in BV2 cells stimulated with 1 μg/mL LPS and 200 μM MGO for 24 h. (g) BV2 cells were electroporated with IκBζ-expression plasmid (IκBζ-plasmid) or control plasmid ( N C-plasmid) and stimulated by 1 μg/mL LPS ± 200 μM MGO for 24 h. Expression levels of IL-1β and iNOS were determined using flow cytometry. (h) BV2 cells were stimulated with 1 μg/mL LPS ± 200 μM MGO for 2 h and treated with doxycycline for the indicated time. The Nfkbiz (IκBζ) mRNAs were determined by real-time PCR. (i) BV2 cells were stimulated with 1 μg/mL LPS ± 200 μM MGO for 24 h, and the expression of Mcpip was analyzed by real-time PCR. (j) BV2 cells were electroporated with firefly luciferase-reporter plasmid (IκBζ-promotor-Luc) and the internal control plasmid (Renilla-Luc) and stimulated with 1 μg/mL LPS ± 200 μM MGO for 24 h. Firefly luciferase activity was determined and normalized to the Renilla luciferase activity. Three independent experiments were performed. * p < 0.05; ** p < 0.01; *** p < 0.001. Ns, not significant.

Article Snippet: Cells were stimulated with 1 μg/mL LPS ( E. coli , Sigma Aldrich) or 10 ng/mL IL-4 (214–14, Peprotech) and 10 ng/mL IL-13 (210–13, Peprotech) in the presence of MGO or not for indicated time.

Techniques: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Flow Cytometry, Luciferase, Activity Assay

Journal: Redox Biology

Article Title: Methylglyoxal suppresses microglia inflammatory response through NRF2-IκBζ pathway

doi: 10.1016/j.redox.2023.102843

Figure Lengend Snippet: MGO suppresses pro-inflammatory cytokine production through the NRF2 pathway. (a) Real-time PCR analysis of Ho1 , Nqo1 , Gclc , and Gclm of BV2 cells stimulated with 1 μg/mL LPS ± 200 μM MGO for 12 and 24 h. (b) Immunofluorescent stain of NRF2 in BV2 cells after treatment with LPS ± 200 μM MGO for 24 h. (c) BV2 cells were electroporated with NRF2-siRNA or N C-siRNA and stimulated with 1 μg/mL LPS ± 200 μM MGO for 24 h. NRF2 knock-down efficiency after NRF2-siRNA electroporation detected by real-time PCR. (d) Expression of IL-1β was addressed by real-time PCR. (e) Expressions of iNOS were determined by flow cytometry. (f and g) Expression of IκBζ was addressed by real-time PCR and Western blot. (h) Putative NRF2-binding site at IκBζ promoter predicted by JASPAR. (i) BV2 cells were stimulated with LPS ± 200 μM MGO for 6 h, and ChIP assays were performed with antibodies against NRF2 or control rabbit IgG. The precipitated DNA containing the IκBζ promoter was assayed by quantitative real-time PCR with specific primers. (j) Schematic diagram showing the mechanism of action of MGO. Representative data from at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. Ns, not significant.

Article Snippet: Cells were stimulated with 1 μg/mL LPS ( E. coli , Sigma Aldrich) or 10 ng/mL IL-4 (214–14, Peprotech) and 10 ng/mL IL-13 (210–13, Peprotech) in the presence of MGO or not for indicated time.

Techniques: Real-time Polymerase Chain Reaction, Staining, Electroporation, Expressing, Flow Cytometry, Western Blot, Binding Assay