Journal: Stem cell research & therapy

Article Title: Intracellular osteopontin potentiates the immunosuppressive activity of mesenchymal stromal cells.

doi: 10.1186/s13287-024-03979-8

Figure Lengend Snippet: Fig. 5 iOPN promoted STAT1-mediated immunosuppression in MSCs. a–d Vector-MSCs and iOPN-MSCs or WT-MSCs and OPN−/−-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL) for the indicated times. Cells were harvested, and OPN, NF-κB p65, STAT1, IKBα, phosphorylation of NF-κB p65, phosphorylation of STAT1 at Tyr701 and phosphorylation of IKBα were analyzed by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 5a-d. e–g Vector-MSCs and iOPN-MSCs were stimulated with TNF-α plus IFN-γ (10 ng/mL) for 12 h. The STAT1 inhibitor fludarabine (Flu, 2 μM) was then added to the culture medium of Vector-MSCs and iOPN-MSCs. The expression of iNOS was determined by quantitative real-time PCR (e), immunoblotting analysis (f) and flow cytometry (g). Full-length blots are presented in Additional file 1: Fig. 5f. h Vector-MSCs and iOPN-MSCs were pretreated with DMSO or fludarabine (Flu, 2 μM) for 6 h and then irradiated and cocultured with CFSE-labeled splenocytes activated by anti-CD3/CD28 antibodies for 3 days at a ratio of 1:20. CD4+ T cells and CD8+ T cells were stained for proliferation analysis by flow cytometry at the end of coculture, and the percentages of proliferating T cells are shown. The results are representative of three to six independent experiments. Values are shown as the mean ± SEM and statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: Antibodies against GAPDH (CAT# 2118, RRID: AB_561053), NF-κB p65 (CAT# 8242, RRID:AB_10859369), phospho-NF-κB p65 (CAT# 3033, RRID:AB_331284), STAT1 (CAT# 14994, RRID:AB_2737027), phospho-STAT1 (Tyr701) (CAT# 7649, RRID:AB_10950970), IκBα (CAT# 4814, RRID:AB_390781), phospho-IκBα (CAT# 2118) and ubiquitin (CAT# 3936, RRID: RRID:AB_331292) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Plasmid Preparation, Phospho-proteomics, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Irradiation, Labeling, Staining

Journal: European journal of immunology

Article Title: β-Glucan enhances antitumor immune responses by regulating differentiation and function of monocytic myeloid-derived suppressor cells.

doi: 10.1002/eji.201242841

Figure Lengend Snippet: Figure 5. WGP-induced differentiation of M-MDSCs is mediated by Syk and NF-κB p65. (A) Sorted splenic MDSCs from tumor-bearing mice were treated with or without WGP (100 μg/mL) for indicated times. Cells were lysed, and western blot analysis developed with anti-phospho-Syk antibody and anti-phospho-NF-κB p65 antibody. β-Actin served as a loading control. (B) M-MDSCs were pretreated for 1 h with or without the NF-κB inhibitors PDTC (100 μM) prior to the addition of WGP. After 48 h incubation, the Gr-1+CD11b+ MDSCs, CD11c+F4/80+ cells were analyzed by flow cytometry. (C) Levels of arginase, nitrite, and IL-12 were measured in the cells or supernatants. Results are represented as mean ± SD of four samples pooled from three independent experiments. Student’s t-test was used for the statistical analysis. **p < 0.01, *p < 0.05.

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto immobilon polyvinylidene fluoride (PVDF) membranes (BioRad, Hercules, CA), and probed with rabbit phospho-Syk antibody (CST, Danvers, MA), rabbit phospho-NF-κB p65 antibody (CST, Danvers, MA), rabbit NF-κB p65 antibody (CST, Danvers, MA) and mouse β-actin antibody (Abcam, Cambridge, UK) followed by chemiluminescent detection (Champion Chemical, Whittier, CA).

Techniques: Western Blot, Control, Incubation, Cytometry

Journal: Annals of the rheumatic diseases

Article Title: Leptin produced by joint white adipose tissue induces cartilage degradation via upregulation and activation of matrix metalloproteinases.

doi: 10.1136/annrheumdis-2011-200372

Figure Lengend Snippet: Figure 2 Signalling pathways activated after leptin and leptin plus IL-1 stimulation of chondrocytes and their role in collagenase expression. Primary human articular chondrocytes were serum starved overnight and then stimulated for 20 and 60 min with either leptin (25 µg/ml) or IL-1 (0.25 ng/ml) alone or in combination. Cells were lysed, and extracts immunoblotted with antibodies against (A) phospho-STAT1 (Y701 or S727), phospho-STAT3 (Y705 and S727), phospho-STAT5 (Y694) and β-tubulin as loading control; (B) phospho-Erk (p44/42) (T202/Y204 of Erk1), phospho-JNK (SAPK) (T183/Y185), phospho-p38 (T180/Y182); or (C) phospho-Akt (S473 and Y308) and phospho-p65 (S536). (D and E) Primary human chondrocytes were stimulated with leptin (25 µg/ml) with or without the inhibitors, LY (LY249002, 5 µM), U0126 (5 µM), SB (SB203580, 10 µM), SP (SP600125, 10 µM), Akt IV (3 µM) or Akt VIII (3 µM) for 20 h. Cells were pretreated for 30 min with inhibitors or vehicle control before the addition of leptin. The expression of MMP1 (D) and MMP13 (E) was assayed by real-time reverse transcription PCR and expressed as fold relative to the control after normalisation to 18s rRNA. *p<0.05; **p<0.01; ***p<0.001 versus control. Data are representative of three separate experiments. IL, interleukin; MMP, matrix metalloproteinase.

Article Snippet: Primary antibodies against phospho-Erk1/2 (phospho-p44/42) (#9101), phospho-p38 (#9211), phosphoJNK1/2 (#9251), phospho-p65 (Ser 536; #3031) and phosphoSTAT were all from Cell Signaling Technology (New England Biolabs, Hitchin, UK).

Techniques: Expressing, Control, Reverse Transcription