Journal: Journal of leukocyte biology

Article Title: Rapid externalization of 27-kDa heat shock protein (HSP27) and atypical cell death in neutrophils treated with the sphingolipid analog drug FTY720.

doi: 10.1189/jlb.3VMA1114-522RR

Figure Lengend Snippet: Figure 5. RIP1K and HSP90 are involved in neutrophil death induced by FTY720. (A and B) Flow cytometric quantification of Annexin V+PI+, Annexin V+PI2, and HSP27+ neutrophils after preincubation or not with necrostatin-1 (20 mM) or Z-IETD-FMK (20 mM) for 30 min and treatment or not with FTY720 (10 mM) for 3 h. Data are from 7 independent experiments. *P , 0.05; **P , 0.01; ***P , 0.001. (C and D) Neutrophil lysates analyzed by Western blotting for caspase-8 cleavage or MLKL phosphorylation after cell death induc- tion by FTY720. Where indicated, Q-VD-OPh (20 mM), necrostatin-1 (20 mM), DPI (20 mM), or NSA (5 mM) was added for 30 min before stimulation. GAPDH analysis was performed as loading control. The results of 1 representative experiment out of 4 are presented. (E) Flow cytometric quantification of HSP27+ neutrophils incubated for 30 min in the presence of geldanamycin (10 mM), DMAG (20 mM), or radicicol (10 mM) before treatment with FTY720 (10 mM) for 3 h. Data are from 7 independent experiments. *P , 0.05; **P , 0.01.

Article Snippet: Selective antagonists of S1PRs [JTE013, VPC23019, W146, or FTY720 (S)-phosphate; all from Santa Cruz Biotechnology] were added to neutrophils for 1 h and washed out before FTY720 treatment.

Techniques: Western Blot, Phospho-proteomics, Control, Incubation

Journal: Cancer Gene Therapy

Article Title: β3-adrenergic receptor on tumor-infiltrating lymphocytes sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma

doi: 10.1038/s41417-023-00599-x

Figure Lengend Snippet: A Representative cytofluorimetric plots and relative quantification of PD-L1 expression on N2A tumor cells treated in vitro with IFN-γ (100 ng/mL), TNF-α (100 ng/mL) in presence or not of SR59230A (1 µM) for 48 h. B Immunofluorescence and relative quantification of PD-L1 staining on N2A tumor cells co-cultured with lymphocytes obtained from TDLNs of mice treated for 2 days with vehicle or SR59230A. C IFN-γ quantification in co-culture medium after 48 h of tumor cells-lymphocytes co-culture. D Representative cytofluorimetric plots and relative quantification of PD-L1 expression on N2A tumor cells co-cultured with lymphocytes obtained from TDLNs of mice treated for 2 days with vehicle or SR59230A. E Representative cytofluorimetric plots and relative quantification of PD-L1 expression on N2A tumor cells co-cultured with lymphocytes silenced for different β-AR subtypes. F Representative images of tumor mass and relative G tumor growth and H tumor weight of NB obtained from A/J mice inoculated with N2A tumor cells, and treated with SR59230A in presence or not of FTY720. Data are expressed as mean ± SEM. A – E ( n = 3 per group); F – H ( n = 6 per group). Significance was calculated by one-way ( A – E , H ) or two-way ( G ) ANOVA analysis followed by Bonferroni’s post hoc test. ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs Vehicle; ## P < 0.01, ### P < 0.001, #### P < 0.0001 N2A + Lymph (SR) vs N2A + Lymph (Veh); $$ P < 0.01, $$$ P < 0.001 SR59230A vs SR59230A + FTY720; ns not significant.

Article Snippet: Treatment with FTY720 (#6176, Tocris Biotechne) was administered at 2 mg/kg per os (p.o.) from day 6.

Techniques: Quantitative Proteomics, Expressing, In Vitro, Immunofluorescence, Staining, Cell Culture, Co-Culture Assay