Journal: Cell reports

Article Title: VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms

doi: 10.1016/j.celrep.2023.113661

Figure Lengend Snippet: WT and Vsir −/− BM progenitor cells were cultured with GM-CSF (10 ng/mL) and IL-6 (10 ng/mL) for 4 days either in normoxia or exposed to hypoxia during the last 24 h. M-MDSCs (Ly6C + Ly6G neg CD11c neg ) were purified and cocultured with OT1 splenocytes (50,000) at the indicated ratios in the presence of the ovalbumin peptide (SIINFEKL, 100 μg/mL). Expanded OT1 T cells were enumerated by flow cytometry after 72 h of stimulation. (A) Normoxic M-MDSCs and OT1 cells were co-cultured under normoxia for 72 h (n = 4 replicates). (B) Hypoxic M-MDSCs and OT1 cells were continuously co-cultured under hypoxia for 72 h (n = 4 replicates). (c) Hypoxic M-MDSCs were co-cultured with OT1 cells under normoxia for 72 h (n = 4 replicates). (D and E) Protein expression of Arg1 and iNOS in normoxic or hypoxic M-MDSCs after 4-day culture was examined by flow cytometry (n = 3 replicates) and by western blotting. (F) Hypoxic WT and Vsir −/− M-MDSCs were rested for 8 h in medium without any cytokines and restimulated with IL-6. Cells were lysed at the indicated time points. Phosphorylated and total levels of STAT3 were detected by western blotting. (G) Hypoxic WT and Vsir −/− M-MDSCs were rested for 8 h and restimulated with GM-CSF. Cell lysates were generated and examined for phosphorylated and total levels of STAT5 and ERK1/2. The ratios of phosphorylated vs. total proteins were quantified using ImageJ. (H and I) Vsir −/− BM progenitor cells were transduced with a retrovirus expressing the mutant STAT3 proteins Y705E.GFP and S727D.GFP as indicated. Cells were expanded in GM-CSF and IL-6 for 4 days, and the expression of Arg1 was examined by flow cytometry. GFP neg cells that did not express the mutant STAT3 are shown as parallel negative controls. All data were presented as mean ± SEM. * p < 0.05; ** p < 0.025; *** p < 0.005; **** p < 0.0001. All experiments were repeated at least three times, and representative results are shown.

Article Snippet: STAT3 inhibitor (Stattic) , Tocris Bioscience Inc , 2798.

Techniques: Cell Culture, Purification, Flow Cytometry, Expressing, Western Blot, Generated, Transduction, Mutagenesis

Journal: Cell reports

Article Title: VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms

doi: 10.1016/j.celrep.2023.113661

Figure Lengend Snippet: (A and B) WT and Vsir −/− BM-derived MDSCs were cultured with GM-CSF and IL-6 for 4 days either in normoxia or exposed to hypoxia during the last 24 h. Total cell lysates were extracted and examined for ODC1 expression (A). Intracellular putrescine levels were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (B) (n = 4 for normoxia replicates, n = 3 for hypoxia replicates). (c) Hypoxic WT and Vsir −/− MDSCs were cultured as in (A) in the presence of putrescine (800 μM) or vehicle control. Cells were rested for 8 h before being restimulated with IL-6. Total cell lysates were examined by western blotting. The ratios of phosphorylated-STAT3 Y705 and STAT3 S727 vs. total STAT3 were quantified using ImageJ. (D) WT BM-derived MDSCs were cultured as in (A) in the presence of DFMO (400 μM) or vehicle control for 4 days. Total cell lysates were generated, and levels of pSTAT3 Y705 and total STAT3 were examined by western blotting. (E) WT BM progenitors (n = 3) were cultured for 4 days as in (A). On day +1, CK2 inhibitor (20 μM), putrescine (800 μM), vehicle control, or combined drugs were added to the culture medium and continuously cultured for another 3 days. During the last 24 h, cells were treated with hypoxia before flow analysis to detect Arg1 expression. (F) WT and Vsir −/− BM-MDSCs (n = 3) were cultured for 4 days as in (A). On day +1, STAT3 inhibitor (2 mM), putrescine (1 mM), vehicle control, or combined drugs were added to the culture medium. Cells were treated with hypoxia during the last 24 h before flow analysis. All experiments were repeated at least three times, and representative results are shown. (G and H) WT and Vsir −/− BM-MDSCs were cultured for 4 days under normoxia or exposed to hypoxia during the last 24 h. Mitochondrial respiration of normoxic (G) and hypoxic MDSCs (H) was examined using the Seahorse XF Cell Mito stress test. The OCR and ECAR were recorded simultaneously. (I) Basal OCR, maximal OCR, SRC, and ECAR are summarized. (J) The bioenergetic profiles of WT and Vsir −/− MDSCs were plotted based on basal OCR and ECAR levels. (K and L) To rescue the defective mitochondrial function in Vsir −/− MDSCs, putrescine (800 μM) was added to the culture medium on day +1. Cells were cultured for a total of 4 days and treated with hypoxia during the last 24 h. M-MDSCs were purified and examined using the Mito stress test. OCR was recorded (K). Basal OCR, maximal OCR, and SRC are summarized (L). (M and N) Total cell lysates were extracted, and the expression of ETC complex V and Arg1 was examined. β-Actin was used as the loading control. The ratios of each protein vs. β-actin were quantified using ImageJ. All experiments were repeated at least three times, and representative results are shown. Statistical analysis: n = 8 (WT, normoxia), 9 (KO, normoxia), 8 (WT, hypoxia), and 10 (KO, hypoxia) replicates (A–C); n = 5 (WT control), 4 (WT putrescine), 14 (KO, control), and 8 (KO putrescine) replicates (D). All data were presented as mean ± SEM. * p < 0.05; ** p < 0.025; *** p < 0.005; **** p < 0.0001.

Article Snippet: STAT3 inhibitor (Stattic) , Tocris Bioscience Inc , 2798.

Techniques: Derivative Assay, Cell Culture, Expressing, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Control, Western Blot, Generated, Purification

Journal: Cell reports

Article Title: VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms

doi: 10.1016/j.celrep.2023.113661

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: STAT3 inhibitor (Stattic) , Tocris Bioscience Inc , 2798.

Techniques: Virus, Western Blot, Recombinant, Staining, cDNA Synthesis, SYBR Green Assay, Isolation, Mutagenesis

Journal: International journal of molecular medicine

Article Title: Curcumin suppresses MUC5AC production via interfering with the EGFR signaling pathway.

doi: 10.3892/ijmm.2018.3609

Figure Lengend Snippet: Figure 4. Effect of curcumin on AKT and STAT3 phosphorylation. H292 cells were treated with curcumin (20 µM) and EGF (50 ng/ml) or vehicle (0.1% dimethylsulfoxide in PBS with 0.1% bovine serum albumin) for different exposure times (10 or 30 min). The phosphorylation of (A) AKT‑Ser473 and (B) STAT3 was determined by western blot analysis. (C and D) H292 cells were treated with LY294002 (25 µM) or curcumin (20 µM) and EGF (50 ng/ml) for 30 min. Nuclear p‑STAT3 was detected by western blot analysis. Results were obtained from 3 separate experiments. Values are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. EGF‑induced model group. EGF, endothelial growth factor; p‑STAT3, phosphorylated signal transducer and activator of transcription 3.

Article Snippet: Cells were treated with curcumin (20 μM at 37 ̊C for 0, 1, 3, 24 and 48 h or 10 and 30 min; 10 μM at 37 ̊C for 48 h; Selleck Chemicals, Houston, TX, USA), EGF (50 ng/ml at 37 ̊C for 0, 3, 24 and 48 h, or 10 and 30 min; PeproTech, Inc., Rocky Hill, NJ, USA), PI3K inhibitor LY294002 (25 μM at 37 ̊C for 30 min or 48 h) and a STAT3 inhibitor STATTIC (10 μM at 37 ̊C for 48 h) (both from Selleck Chemicals).

Techniques: Phospho-proteomics, Western Blot, Standard Deviation, Control

Journal: Allergy

Article Title: Epigenetic regulation of epithelial dectin-1 through an IL-33-STAT3 axis in allergic disease

doi: 10.1111/all.14898

Figure Lengend Snippet: (a) representative flow plots and (b) frequencies of STAT3 pTyr705 phosphoflow in CRSwNP hSNECs treated with IL-33 for 2h. CLEC7A promoter activity in (c) bronchial epithelial cell line or (d) Clec7a mRNA in isolated mouse tracheal epithelial cells from Stat3+/+ and Stat3−/− mice. (e) STAT3 binding at the CLEC7A locus in bronchial epithelial cell line treated with media or IL-33 (10 ng/ml) for 2h. CLEC7A mRNA in CRSwNP SNECs treated with (f) HDM (100 ug/ml) or (g) IL-33 (0.25 ng/ml) and vehicle or 5 uM STAT3 inhibitor, stattic for 2h. Data is means+SEM and representative of 2–3 independent experiments, representative of 3 donors (a,b,f,g). Data was analyzed by one-way ANOVA followed by post-hoc test or two-way ANOVA followed by Bonferroni’s multiple comparisons test (panel e, where significances are displayed for IL-33 (anti-STAT3) vs. IL-33 (anti-GFP)). *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

Article Snippet: Where indicated, cells were pretreated for 2h with 5 uM stattic (Cayman) or DMSO, or 5 ug/ml isotype or neutralizing monoclonal anti-hIL-33 antibodies (R&D Systems, MAB36254), followed by stimulation with rIL-33 (Peprotech), HDM (100 ug/ml) for 2h.

Techniques: Activity Assay, Isolation, Binding Assay

Journal: Journal of neurochemistry

Article Title: Phosphatidylinositol-3 kinase mediates the sweet suppressive effect of leptin in mouse taste cells

doi: 10.1111/jnc.15268

Figure Lengend Snippet: Effect of bath applied leptin on sucrose responses of taste receptor family 1 member 3 (T1R3)-green fluorescent protein (GFP) taste cells (TCs) from mouse fungiform papillae. (a) Sample recordings of TC responses to 500 mM sucrose (Suc) before (cont), during (leptin) and after (wash) treatment with 20 ng/ml leptin. The picture shows a T1R3-GFP TC from which taste responses were recorded. (b). Responses of 10 individual T1R3-GFP TCs to 500 mM sucrose before (left), during (middle) and after (right) treatment of 20 ng/ml leptin. (c) Effects of leptin on summated responses of T1R3-GFP TCs to 500 mM sucrose (n = 10 cells/ 9 mice, F = 14.869, p < .001, repeated ANOVA). Values are means ± SE. (d–i) Phosphoinositide 3-kinase (PI3K) inhibitors suppressed leptin’s suppression of TC responses to sucrose. (d, g) Sample recordings of TC responses to 500 mM sucrose (Suc) before (cont), during (leptin) and after (wash) treatment with 20 ng/ml leptin. PI3K inhibitor wortmannin (30 nM, d) or LY294002 (30 μM, g) was added to bath solution throughout the experiment. The picture shows a T1R3-GFP TC from which taste responses were recorded. (e, h) Responses of 8 and 7 individual T1R3-GFP TCs to 500 mM sucrose before (left), during (middle) and after (right) treatment of 20 ng/ml leptin under the existence of 30 nM wortmannin (e) or 30 μM LY294002 (h). (f, i) The effects of leptin on summated responses of T1R3-GFP TCs to 500 mM sucrose under the existence of 30 nM wortmannin (n = 8 cells/ 7 mice, F = 0.3577, p > .1, repeated ANOVA, f) or 30 μM LY294002 (n = 7 cells/ 7 mice, F = 0.0375, p > .1, repeated ANOVA, i). Values are means ± SE. (j–o) Signal transducer and activator of transcription 3 (STAT3) inhibitor and Src homology region 2 domain-containing phosphatase-2 (SHP2) inhibitor did not affect leptin’s suppression of TC responses to sucrose. (j, m) Sample recordings of TC responses to 500 mM sucrose (Suc) before (cont), during (leptin) and after (wash) treatment with 20 ng/ml leptin. STAT3 inhibitor stattic (10 μM, i) or SHP2 inhibitor SHP099 (10 μM, m) was added to bath solution throughout the experiment. The picture shows a T1R3-GFP TC from which taste responses were recorded. (k, n) Responses of 8 individual T1R3-GFP TCs to 500 mM sucrose before (left), during (middle) and after (right) treatment of 20 ng/ml leptin under the existence of 10 μM stattic (k) or 10 μM SHP099 (n). (l, o) Effects of leptin on summated responses of T1R3-GFP TCs to 500 mM sucrose under the existence of 10 μM stattic (n = 8 cells/ 8 mice, F = 4.0694, p < .05, repeated ANOVA, l) or 10 μM SHP099 (n = 8 cells/ 7 mice, F = 5.8569, p < .05, repeated ANOVA, o). Values are means ± SE. **p < .01, *p < .05, post hoc paired t-test with Holm correction

Article Snippet: Recombinant murine leptin (20 ng/ml; PeproTech, Cat# 450-31), wortmannin (10 nM; Tocris Bioscience, Cat# 1,232), LY294002 (30 μM; Sigma-Aldrich, Cat# L9908), SHP099 (10 μM; Cayman Chemical, Cat# 20,000), and stattic (10 μM; Axon Medchem, Cat# 2,314) were applied from the basolateral side of TCs.

Techniques:

Journal: International Journal of Oncology

Article Title: STAT3 activation in HER2-overexpressing breast cancer promotes epithelial-mesenchymal transition and cancer stem cell traits

doi: 10.3892/ijo.2013.2195

Figure Lengend Snippet: STAT3 inhibitor, Stattic abolishes the stem cell marker expression. (A) Dose response curve of MCF7-HER2 for the STAT3 inhibitor Stattic. (B) Stattic treatment abolished pSTAT3 and the stem cell marker expression in MCF7-HER2. The EMT driver slug expression was also abolished upon Stattic treatment. (C) Western blot analyses showed that, with Stattic treatment, expression of vimentin and slug was downregulated while E-cadherin expression was upregulated in MCF7-HER2. (D) Combined treatment of Herceptin and Stattic showed a synergistic cell growth inhibition effect on HER2-overexpressing, ER-positive breast cancer cells.

Article Snippet: Cells were allowed to grow until about 50% confluence, and then treated with the following dosage treatments: untreated control, Stattic (5 μ M), Herceptin (10 μ g/ml, a gift of Genentech) and Stattic (5 μ M) + Herceptin (10 μ g/ml) combination.

Techniques: Marker, Expressing, Western Blot, Inhibition