Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 maintains BTIC self-renewal. ( a ) Morphology of self-renewing spheres and immunofluorescence of neuronal and glial lineage markers in differentiated cells. Scale bar=200 μm. ( b ) Immunofluorescence of ING5 in undifferentiated (upper panels) and cells differentiated for 5 days (lower panels). Scale bar=20 μm. ( c , d ) The mRNA levels ( c ) and protein levels ( d ) of ING5 decrease during differentiation in BT 189 cells. n =4. ( e ) Immunoblotting of ING5 protein in the BT 12, and BT 134 lines during differentiation. ( f ) ING5 overexpression increases sphere formation rates and average volumes in BTIC sphere formation assays ( n =3, ** P <0.01, * P <0.05). Scale bar=400 μm. ( g ) ING5 knockdown by siRNA decreases sphere formation rates and sphere sizes of BTICs ( n =4, ** P <0.01, * P <0.05). Scale bars=400 μm. ( h ) (Left) Sphere formation rates for cell lines stably expressing shRNAs against ING5 (shR1 and shR2-ING5) or control non-targeting shRNA (shR-ctr). ( n =4, ** P <0.01, * P <0.05). (Right) Fluorescence of the RFP reporter in stable cell lines superimposed with differential interference contrast (DIC) images. Scale bar=100 μm. ( i ) RT-qPCR analysis of stem cell core transcription factors and stem cell markers after ING5 overexpression. ( j , k ) Western blot analysis of the neural stem cell marker Nestin and neuronal lineage marker Tubb3 in response to ING5 overexpression ( j ) and knockdown ( k ).

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Western Blot, Over Expression, Knockdown, Stable Transfection, Expressing, Control, shRNA, Fluorescence, Quantitative RT-PCR, Marker

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 increases the stem cell pool and inhibits differentiation. ( a ) Flow cytometry analysis of CD133/CD44 positive cells in iPB cell lines (top panels) and CD44 positive cells in shRNA cell lines (bottom panels), gated by isotype control. ( b ) (Left) Mitotic pair analysis of the three division modes: symmetric proliferating (sym-pro), symmetric differentiating (sym-diff) and asymmetric (asym) cell division, in iPB cell lines. Over 150 pairs were counted for each group in one experiment. n =3, * P <0.05. (Right) An example of cell division symmetry based on the distribution of stem cell factor Nestin with the Red arrow indicating symmetric differentiating division and the white arrow asymmetric division. ( c ) Morphological changes of iPB cells before (Day 0) and after (Days 1–3) differentiation induced by 1% FBS. Scale bar=100 μm. ( d ) After differentiation for 5 days, immunofluorescence of Nestin and Tubb3 in shRNA cell lines with an RFP reporter. Scale bar=200 μm. ( e ) Western blot of Nestin and Tubb3 in differentiated shRNA cell lines corresponding to cells shown in d .

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Flow Cytometry, shRNA, Control, Immunofluorescence, Western Blot

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 activates mitogenic pathways to promote self-renewal. ( a ) The sphere formation rates of iPB-ctr and iPB-ING5 overexpressing cells at three successive passages in the absence of EGF and FGF treatment. ( n =3, ** P <0.01, *** P <0.001). ( b ) DIC images of spheres from the tertiary sphere passage in iPB cells. Scale bar=400 μm. ( c ) Sphere formation rate under treatment with protein kinase inhibitors ( n =3, * P <0.05). ( d ) Protein and phosphorylated protein levels of effectors in the PI3K and MEK pathways. Cells were treated with PX-866 at 1 μ M and PD184352 at 2 μ M for 48 h. ( e ) Immunofluorescence of Nestin and Tubb3 in differentiated iPB control (Left panels) or iPB-ING5 cells (Right panels) treated with 1 μ M PX-866 or 2 μ M PD184352. Scale bar=200 μm. ( f ) Flow cytometry analysis of the CD133 positive population in PX-866 (1 μ M ) and PD184352 (2 μ M ) treated BTIC 189 cells, gated by isotype control.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Control, Flow Cytometry

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: The FSH pathway transduces effects of ING5 on stem cell properties. ( a ) Sphere formation assay for cells treated with calcium modulators and FSHR blocking antibody (Anti-FSHR) at the indicated concentrations ( n =3, * P <0.05 and ** P <0.01 compared to iPB-ctr/DMSO; # P <0.05 and ## P <0.01 compared to iPB-ING5/DMSO). ( b ) IPA downstream function analysis indicates the FSH pathway is elevated by ING5. Genes positively correlated with this function were listed with fold changes. ( c ) RT-qPCR of genes related to hormone and steroidogenesis functions. ( n =3, * P <0.05, ** P <0.01) ( d ) The expression levels of FSHB and FSHR genes in BT 189 cells before and after differentiation for 1–5 days. ( e ) Immunostaining for ING5 and FSHR in iPB cells. Scale bar=100 μm. ( f ) Flow cytometry analysis of CD133 positive cells in BT 189 cells treated with FSHR neutralizing antibody or IgG control, gated by isotype control. ( g ) Immunofluorescence of Nestin and Tubb3 shows inhibition of the FSH pathway induces neuronal differentiation. Scale bar=200 μm. ( h ) FSH recombinant protein treatment at indicated concentrations increases sphere formation rates in shRNA cell lines ( n =3, * P <0.05 and ** P <0.01 compared to untreated shR-ctr; # P <0.05 compared to untreated shR-ING cells). ( i ) FSH recombinant protein treatment induces sphere-forming abilities in iPB-ctr cells but not in ING5 overexpressing cells ( n =3, ** P <0.01). ( j ) Sphere formation rates for cells treated with Anti-FSHR or BAPTA alone, and the combination of both ( n =3, * P <0.05). ( k ) FSH treatment at 5 ng/ml for 3 days induces the expression of OCT4 and Nestin in BT 189 cells.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Tube Formation Assay, Blocking Assay, Quantitative RT-PCR, Expressing, Immunostaining, Flow Cytometry, Control, Immunofluorescence, Inhibition, Recombinant, shRNA

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: PHD motif is required for the function of ING5 in BTICs and ING5 levels negatively correlate with survival of GBM. ( a ) Sphere formation assays in iPB cell lines overexpressing wild-type (ING5-FLAG) and PHD-deleted ING5 (ΔPHD) ( n =3, ** P <0.01). ( b ) Western blot shows the protein levels of endogeneous ING5, wildtype ING5 with a FLAG tag and PHD-deleted ING5 (black arrows) in three iPB cell lines. ( c ) ChIP analysis of ING5 binding to promoters of target genes presented as fold enrichment relative to IgG controls. The endogenous ING5 in BT 189 cells, overexpressed ING5 with a Flag tag in iPB-ING5 cells and overexpressed PHD-deleted ING5 protein with a Flag tag were immunoprecipitated by the ING5 antibody and Flag antibody respectively. The upper panels are the schematic representation of the location of the primer sets and promoter regions enriched for ING5 binding were shown in red. ( d ) Kaplan–Meier survival analysis of TCGA GBM patients with high and low levels of ING5 expression (stratified by mean value, n =114). ( e , f ) ING5 expression levels negatively correlate with survival of the Proneural subtype ( n =24) and the Classical subtype ( n =30) of GBM patients. ( g ) The relationship of ING5 levels to survival in the SOX2-low group of patients (ING5, SOX2 stratified by median values, n =61). ( h ) Model for how ING5 functions in the maintenance of BTIC self-renewal. In the absence of growth factors, ING5 induces FSH and calcium signaling by promoting transcription of the FSH receptor and ligand genes, and various plasma membrane calcium channel genes. The FSH and calcium signaling pathways further activate PI3K/AKT and MEK/ERK signaling to induce stem cell features and the expression of stemness factors OCT4, OLIG2 and Nestin. Gene activation by ING5 is dependent on its PHD motif to target ING5-associated histone acetyltransferase complexes to the promoters.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Western Blot, FLAG-tag, Binding Assay, Immunoprecipitation, Expressing, Clinical Proteomics, Membrane, Protein-Protein interactions, Activation Assay

Journal: Frontiers in Endocrinology

Article Title: Low glucose availability potentiates the effects of metformin on model T cell activation and exhaustion markers in vitro

doi: 10.3389/fendo.2023.1216193

Figure Lengend Snippet: The effect of metformin as a function of glucose availability on CD4+ T cell differentiation in PBMC. PBMC were treated for 72h with metformin in media supplemented with 25 mM, 5.6 mM or 0.56 mM glucose (denoted by shades of red). AMPK activator A 769662 and 4.8 mM 2DG were used as additional controls. After treatment, the cells were stained with the appropriate antibodies and analyzed by flow cytometry. The CD3+ CD4+ population was divided into four populations according to CD45RA and CCR7 (CD197) expression: naïve T cells (Tnaive, CD45RA+ CCR7+, (A) ), central memory T cells (Tcm, CD45RA- CCR7+, (B) ), effector memory T cells (Tem, CD45RA- CCR7-, (C) ) and terminally differentiated effector T cells (Temra, CD45RA+ CCR7-, (D) ). The gating strategy is shown in section 1.2.4 of the Supplementary Material . The percentage of total CD4+ CD3+ cells is shown for each subpopulation. The data points represent individual experiments, while the horizontal lines and error bars represent the median percentage ± interquartile range. Three independent experiments were performed for each of the four healthy donors for a total of twelve independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by repeated measures two-way ANOVA with Dunnett (glucose) or Šidak (metformin) post-hoc test. No significant interaction between metformin and glucose level could be confirmed by repeated measures two-way ANOVA. The distribution of subpopulations according to differentiation status is summarized in (E) by displaying mean percentages of the subsets.

Article Snippet: PD-L1, programmed death-ligand 1; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; IFN-γ, interferon gamma; PD-1, programmed death 1; 2DG, 2-deoxy-D-glucose; CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cells; ATCC, American Type Culture Collection; ATP, adenosine triphosphate; AMPK, adenosine monophosphate activated protein kinase; CD, cluster of differentiation; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; CFSE, carboxyfluorescein succinimidyl ester; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; OxPhos, oxidative phosphorylation; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; IL-2, interleukin 2; NAO, nonyl-acridine orange; ANOVA, analysis of variance; SEM, standard error of the mean; CCR7, C-C chemokine receptor type 7.

Techniques: Cell Differentiation, Staining, Flow Cytometry, Expressing

Journal: Frontiers in Endocrinology

Article Title: Low glucose availability potentiates the effects of metformin on model T cell activation and exhaustion markers in vitro

doi: 10.3389/fendo.2023.1216193

Figure Lengend Snippet: The effect of metformin as a function of glucose availability on CD8+ T cell differentiation in PBMC. PBMC were treated for 72h with metformin in media supplemented with 25 mM, 5.6 mM or 0.56 mM glucose (denoted by shades of red). AMPK activator A 769662 and 4.8 mM 2DG were used as additional controls. After treatment, the cells were stained with the appropriate antibodies and analyzed by flow cytometry. The CD3+ CD8+ population was divided into four populations according to CD45RA and CCR7 (CD197) expression: naïve T cells (Tnaive, CD45RA+ CCR7+, (A) ), central memory T cells (Tcm, CD45RA- CCR7+, (B) ), effector memory T cells (Tem, CD45RA- CCR7-, (C) ) and terminally differentiated effector T cells (Temra, CD45RA+ CCR7-, (D) ). The gating strategy is shown in section 1.2.4 of the Supplementary Material . The percentage of total CD8+ CD3+ cells is shown for each subpopulation. The data points represent individual experiments, while the horizontal lines and error bars represent the median percentage ± interquartile range. Three independent experiments were performed for each of the four healthy donors for a total of twelve independent experiments. *p<0.05, ***p<0.001, ****p<0.0001 as determined by repeated measures two-way ANOVA with Dunnett (glucose) or Šidak (metformin) post-hoc test. No significant interaction between metformin and glucose level could be confirmed by repeated measures two-way ANOVA. The distribution of subpopulations according to differentiation status is summarized in (E) by displaying mean percentages of the subsets.

Article Snippet: PD-L1, programmed death-ligand 1; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; IFN-γ, interferon gamma; PD-1, programmed death 1; 2DG, 2-deoxy-D-glucose; CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cells; ATCC, American Type Culture Collection; ATP, adenosine triphosphate; AMPK, adenosine monophosphate activated protein kinase; CD, cluster of differentiation; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; CFSE, carboxyfluorescein succinimidyl ester; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; OxPhos, oxidative phosphorylation; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; IL-2, interleukin 2; NAO, nonyl-acridine orange; ANOVA, analysis of variance; SEM, standard error of the mean; CCR7, C-C chemokine receptor type 7.

Techniques: Cell Differentiation, Staining, Flow Cytometry, Expressing

Journal: Frontiers in Endocrinology

Article Title: Low glucose availability potentiates the effects of metformin on model T cell activation and exhaustion markers in vitro

doi: 10.3389/fendo.2023.1216193

Figure Lengend Snippet: The effect of the metformin treatment on energy metabolism of T cells from PBMC. (A–F) PBMC cells were seeded in media with 5.6 mM glucose and treated for 48h with 0 mM or 5 mM metformin as indicated. (G–L) T cells from PBMC were activated with anti-CD3/anti-CD28 antibodies and treated with 0 mM or 5 mM metformin in media with 5.6 mM or 0.56 mM glucose for 48h as indicated. Baseline OCR (A, G) and ECAR (C, H) as well as maximal OCR (C, I) were determined using Seahorse Mito Stress Test assay with 1.5 μM oligomicin, 2 μM FCCP and 0.5 μM rotenone/antimycin A injections. The ATP production from oxidative phosphorylation (D, J) and glycolysis (E, K) , as well as total ATP production (F, L) were calculated according to the manufacturer’s instructions for Seahorse Real Time ATP Assay. Mean ± SEM is shown for three independent experiments with different donors. *p<0.05 as determined by paired t-test (A–F) or two-way ANOVA with Šidak post-hoc test (G–L) . A representative time-course for OCR is shown in (M) .

Article Snippet: PD-L1, programmed death-ligand 1; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; IFN-γ, interferon gamma; PD-1, programmed death 1; 2DG, 2-deoxy-D-glucose; CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cells; ATCC, American Type Culture Collection; ATP, adenosine triphosphate; AMPK, adenosine monophosphate activated protein kinase; CD, cluster of differentiation; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; CFSE, carboxyfluorescein succinimidyl ester; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; OxPhos, oxidative phosphorylation; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; IL-2, interleukin 2; NAO, nonyl-acridine orange; ANOVA, analysis of variance; SEM, standard error of the mean; CCR7, C-C chemokine receptor type 7.

Techniques: Phospho-proteomics, ATP Assay

Journal: Frontiers in Endocrinology

Article Title: Low glucose availability potentiates the effects of metformin on model T cell activation and exhaustion markers in vitro

doi: 10.3389/fendo.2023.1216193

Figure Lengend Snippet: The effect of metformin as a function of glucose availability on T cell activation and PD-1 expression in activated T cells from PBMC. PBMC were treated for 72h with metformin in media supplemented with 25 mM, 5.6 mM or 0.56 mM glucose (denoted by shades of red) and activated with anti-CD3 and anti-CD28 antibodies during the treatment. After treatment, the cells were stained with antibodies and analyzed by flow cytometry. The expression levels of activation marker CD69 (A, B) , exhaustion marker/immune checkpoint PD-1 (C, D) and the ratio of PD-1/CD69 fluorescence (E, F) were determined in CD4+ (A, C, E) and CD8+ (B, D, F) T cells. The gating strategy is shown in section 1.2.5 of the Supplementary Material . The data points represent individual experiments, while the horizontal lines and error bars represent the median percentage ± interquartile range. Three independent experiments were performed for each of the four healthy donors for a total of twelve independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by repeated measures two-way ANOVA with Dunnett (glucose) or Šidak (metformin) post-hoc test. Repeated measures two-way ANOVA confirmed the significant interaction between metformin and glucose level for CD69 (A, B) and PD-1 (C, D) expression, but not the PD-1/CD69 ratio (E, F) .

Article Snippet: PD-L1, programmed death-ligand 1; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; IFN-γ, interferon gamma; PD-1, programmed death 1; 2DG, 2-deoxy-D-glucose; CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cells; ATCC, American Type Culture Collection; ATP, adenosine triphosphate; AMPK, adenosine monophosphate activated protein kinase; CD, cluster of differentiation; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; CFSE, carboxyfluorescein succinimidyl ester; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; OxPhos, oxidative phosphorylation; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; IL-2, interleukin 2; NAO, nonyl-acridine orange; ANOVA, analysis of variance; SEM, standard error of the mean; CCR7, C-C chemokine receptor type 7.

Techniques: Activation Assay, Expressing, Staining, Flow Cytometry, Marker, Fluorescence

Journal: Frontiers in Endocrinology

Article Title: Low glucose availability potentiates the effects of metformin on model T cell activation and exhaustion markers in vitro

doi: 10.3389/fendo.2023.1216193

Figure Lengend Snippet: The effect of metformin as a function of glucose availability on proliferation, cytokine secretion and transcription factors in activated T cells from PBMC. PBMC were treated for 72h (A–D) or 48h (E–L) with 0.3 mM or 5 mM metformin as indicated in media supplemented with 25 mM, 5.6 mM or 0.56 mM glucose (denoted by shades of red) and activated with anti-CD3 and anti-CD28 antibodies during the treatment. (A, B) The percentage of proliferated PBMC cells following T cells activation was determined by measuring CFSE dilution by flow cytometry. A representative histogram of CFSE fluorescence is shown in (B) . (C, D) The concentration of secreted IL-2 after 24h treatment (C) and IFN-γ in after 72h treatment (D) in culture media was determined with ELISA. (E–L) The expression of Eomes (E, F) , T-bet (G, H) , STAT3 (I, J) and HIF-1α (K, L) was measured by intracellular staining flow cytometry. The gating strategy is shown in section 1.2.6 of the Supplementary Material . (A–D) The data points represent individual experiments, while the horizontal lines and error bars represent the median percentage ± interquartile range. Three independent experiments were performed for each of the four healthy donors for a total of twelve independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by repeated measures two-way ANOVA with Dunnett (glucose) or Šidak (metformin) post-hoc test. Repeated measures two-way ANOVA confirmed the significant interaction between metformin and glucose level for IFN-γ secretion only (D) . (E–L) The data points represent individual experiments, while the horizontal lines and error bars represent the mean ± SEM. One independent experiment was performed for each of three to four donors. *p<0.05, ***p<0.001, as determined by mixed effects model (E–H) or two-way ANOVA (I–L) with Dunnett (metformin) or Šidak (glucose) post-hoc test.

Article Snippet: PD-L1, programmed death-ligand 1; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; IFN-γ, interferon gamma; PD-1, programmed death 1; 2DG, 2-deoxy-D-glucose; CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cells; ATCC, American Type Culture Collection; ATP, adenosine triphosphate; AMPK, adenosine monophosphate activated protein kinase; CD, cluster of differentiation; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; CFSE, carboxyfluorescein succinimidyl ester; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; OxPhos, oxidative phosphorylation; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; IL-2, interleukin 2; NAO, nonyl-acridine orange; ANOVA, analysis of variance; SEM, standard error of the mean; CCR7, C-C chemokine receptor type 7.

Techniques: Activation Assay, Flow Cytometry, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Staining

Journal: Frontiers in Endocrinology

Article Title: Low glucose availability potentiates the effects of metformin on model T cell activation and exhaustion markers in vitro

doi: 10.3389/fendo.2023.1216193

Figure Lengend Snippet: The effect of metformin as a function of glucose availability on mTOR signaling and the role of AMPK in activated T cells from PBMC. (A–F) PBMC were pretreated with 5 μM compound C for 30 min and subsequently treated for 48h with 5 mM metformin as indicated in media supplemented with 5.6 mM or 0.56 mM glucose and activated with anti-CD3 and anti-CD28 antibodies during the treatment. The expression of Eomes (A, B) , T-bet (C, D) and STAT3 (E, F) was measured by intracellular staining flow cytometry. (G, H) PBMC were treated for 48h with 0.3 mM or 5 mM metformin as indicated in media supplemented with 5.6 mM or 0.56 mM glucose (denoted by shades of red) and activated with anti-CD3 and anti-CD28 antibodies during the treatment. The expression of phosphorylated S6RP was measured by intracellular staining flow cytometry. The gating strategy is shown in section 1.2.6 of the Supplementary Material . The data points represent individual experiments, while the horizontal lines and error bars represent the mean percentage ± SEM. One independent experiment was performed for each of three to four donors. *p<0.05, **p<0.01, as determined by mixed effects model (A–D, G, H) or two-way ANOVA (E, F) with Dunnett or Šidak post-hoc test.

Article Snippet: PD-L1, programmed death-ligand 1; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; IFN-γ, interferon gamma; PD-1, programmed death 1; 2DG, 2-deoxy-D-glucose; CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cells; ATCC, American Type Culture Collection; ATP, adenosine triphosphate; AMPK, adenosine monophosphate activated protein kinase; CD, cluster of differentiation; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; CFSE, carboxyfluorescein succinimidyl ester; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; OxPhos, oxidative phosphorylation; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; IL-2, interleukin 2; NAO, nonyl-acridine orange; ANOVA, analysis of variance; SEM, standard error of the mean; CCR7, C-C chemokine receptor type 7.

Techniques: Expressing, Staining, Flow Cytometry

Journal: Frontiers in Endocrinology

Article Title: Low glucose availability potentiates the effects of metformin on model T cell activation and exhaustion markers in vitro

doi: 10.3389/fendo.2023.1216193

Figure Lengend Snippet: Summary of the effects of metformin on Jurkat cells and activated T cells from PBMC in normal/high and low glucose. Red arrows with white filling indicate the effect of metformin in Jurkat cell while full red arrows indicate the effect of metformin in T cells from PBMC. Double red arrows indicate synergistic effects of metformin and low glucose in T cells from PBMC.

Article Snippet: PD-L1, programmed death-ligand 1; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; IFN-γ, interferon gamma; PD-1, programmed death 1; 2DG, 2-deoxy-D-glucose; CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cells; ATCC, American Type Culture Collection; ATP, adenosine triphosphate; AMPK, adenosine monophosphate activated protein kinase; CD, cluster of differentiation; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; CFSE, carboxyfluorescein succinimidyl ester; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; OxPhos, oxidative phosphorylation; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; IL-2, interleukin 2; NAO, nonyl-acridine orange; ANOVA, analysis of variance; SEM, standard error of the mean; CCR7, C-C chemokine receptor type 7.

Techniques:

Journal: Journal of Inflammation Research

Article Title: TXNIP Participated in NLRP3-Mediated Inflammation in a Rat Model of Cervical Spondylotic Myelopathy

doi: 10.2147/JIR.S373614

Figure Lengend Snippet: The expression of TNXIP in the lesioned spinal cord following compression was observed. ( A ) The TNXIP/NeuN expression in the anterior horn of lesioned spinal cords was analyzed by immunofluorescence. The mean fluorescence intensities of TXNIP were analyzed by Image J software, and represented as mean gray values. ( B ) The TXNIP expression in the lesioned spinal cords was analyzed by Western blot. The gray values of protein bands were analyzed by Image J software, and protein expression was normalized to GAPDH. *P<0.05, **P<0.01.

Article Snippet: The primary antibodies included rabbit anti-rat TXNIP (1:800, MA5-32771, ThermoFisher), NLRP3 (1:800, MA5-32255, ThermoFisher), pro-caspase-1 (1:800, MA5-35833, ThermoFisher) and GAPDH (1:10000, PA1-16777, ThermoFisher).

Techniques: Expressing, Immunofluorescence, Fluorescence, Software, Western Blot

Journal: Journal of Inflammation Research

Article Title: TXNIP Participated in NLRP3-Mediated Inflammation in a Rat Model of Cervical Spondylotic Myelopathy

doi: 10.2147/JIR.S373614

Figure Lengend Snippet: Inhibition of TXNIP improved CSM-induced behavioral deficits. ( A ) The21-point Basso, Beattie and Bresnahan (BBB) locomotor rating scale; ( B ) cold allodynia; ( C ) inclined plane test. Compared with the sham group, **P<0.01; compared with the scrambled group, # P<0.05, ## P<0.01.

Article Snippet: The primary antibodies included rabbit anti-rat TXNIP (1:800, MA5-32771, ThermoFisher), NLRP3 (1:800, MA5-32255, ThermoFisher), pro-caspase-1 (1:800, MA5-35833, ThermoFisher) and GAPDH (1:10000, PA1-16777, ThermoFisher).

Techniques: Inhibition

Journal: Journal of Inflammation Research

Article Title: TXNIP Participated in NLRP3-Mediated Inflammation in a Rat Model of Cervical Spondylotic Myelopathy

doi: 10.2147/JIR.S373614

Figure Lengend Snippet: Inhibition of TXNIP reduced apoptosis in the anterior horn of the lesioned area following CSM. ( A ) Positive TUNEL staining control. ( B ) Apoptosis in the anterior horn of the lesioned area was analyzed by TUNEL staining. Black arrows show apoptotic cells. Apoptosis rate (%) = the numbers of apoptosis cells/total numbers of cells ×100%. The numbers of cells were counted using Image J software. Compared with the sham group, **P<0.01; compared with the scrambled group, ## P<0.01.

Article Snippet: The primary antibodies included rabbit anti-rat TXNIP (1:800, MA5-32771, ThermoFisher), NLRP3 (1:800, MA5-32255, ThermoFisher), pro-caspase-1 (1:800, MA5-35833, ThermoFisher) and GAPDH (1:10000, PA1-16777, ThermoFisher).

Techniques: Inhibition, TUNEL Assay, Staining, Control, Software

Journal: Journal of Inflammation Research

Article Title: TXNIP Participated in NLRP3-Mediated Inflammation in a Rat Model of Cervical Spondylotic Myelopathy

doi: 10.2147/JIR.S373614

Figure Lengend Snippet: Inhibition of TXNIP declined the TXNIP expression in the anterior horn of the lesioned area following CSM. The mean fluorescence intensities of TXNIP/NeuN were analyzed by immunofluorescence. The mean gray values were analyzed using Image J software. Compared with the sham group, *P<0.05, **P<0.01; compared with the scrambled group, ## P<0.01.

Article Snippet: The primary antibodies included rabbit anti-rat TXNIP (1:800, MA5-32771, ThermoFisher), NLRP3 (1:800, MA5-32255, ThermoFisher), pro-caspase-1 (1:800, MA5-35833, ThermoFisher) and GAPDH (1:10000, PA1-16777, ThermoFisher).

Techniques: Inhibition, Expressing, Fluorescence, Immunofluorescence, Software

Journal: Journal of Inflammation Research

Article Title: TXNIP Participated in NLRP3-Mediated Inflammation in a Rat Model of Cervical Spondylotic Myelopathy

doi: 10.2147/JIR.S373614

Figure Lengend Snippet: Inhibition of TXNIP declined the NLRP3 expression in the anterior horn of the lesioned area following CSM. The mean fluorescence intensities of NLRP3/NeuN were analyzed by immunofluorescence. The mean gray values were analyzed by Image J software. Compared with the sham group, **P<0.01; compared with the scrambled group, ## P<0.01.

Article Snippet: The primary antibodies included rabbit anti-rat TXNIP (1:800, MA5-32771, ThermoFisher), NLRP3 (1:800, MA5-32255, ThermoFisher), pro-caspase-1 (1:800, MA5-35833, ThermoFisher) and GAPDH (1:10000, PA1-16777, ThermoFisher).

Techniques: Inhibition, Expressing, Fluorescence, Immunofluorescence, Software

Journal: Journal of Inflammation Research

Article Title: TXNIP Participated in NLRP3-Mediated Inflammation in a Rat Model of Cervical Spondylotic Myelopathy

doi: 10.2147/JIR.S373614

Figure Lengend Snippet: Inhibition TXNIP declined the NLRP3 mediated pro-caspase-1 expression in the anterior horn of the lesioned area following CSM. ( A ) The mean fluorescence intensities of pro-caspase-1/NeuN were analyzed by immunofluorescence. ( B ) The expression levels of TXNIP, NLRP3 and pro-caspase-1 in the lesioned spinal cords were measured by Western blot. The mean grays were analyzed by the Image J software. Compared with the sham group, **P<0.01; compared with the scrambled group, ## P<0.01.

Article Snippet: The primary antibodies included rabbit anti-rat TXNIP (1:800, MA5-32771, ThermoFisher), NLRP3 (1:800, MA5-32255, ThermoFisher), pro-caspase-1 (1:800, MA5-35833, ThermoFisher) and GAPDH (1:10000, PA1-16777, ThermoFisher).

Techniques: Inhibition, Expressing, Fluorescence, Immunofluorescence, Western Blot, Software