Journal: Cell Death Discovery

Article Title: Preventing light-induced toxicity in a new mouse model of sector retinitis pigmentosa caused by Rhodopsin M39R variant

doi: 10.1038/s41420-025-02769-2

Figure Lengend Snippet: A Transcriptomic analysis by bulk RNA-seq of Rho M39R/M39R KI retinae compared to control mice. The volcano plot shows the top 35 differentially expressed genes by adjusted p value. S1P signalling pathway genes are underlined in red. fc_only = only fold change; not_sign = not significant; sig_only = only significant; sig+fc = significant plus fold change. B Rho M39R/+ KI mice were treated with FTY720 at 4 weeks of age. Schematic of the treatment in Rho M39R/+ KI mice. The animals were dark-adapted overnight and intraperitoneally injected with 10 mg/kg of FTY720 or vehicle (saline solution) 30 min before performing the light damage assay. The light damage consisted in performing an ERG every week for 4 times in total. Each time, the mice were preinjected with FTY720 or vehicle. C The ONL thickness was measured at day 21, after 4 rounds of ERG. FTY720-treated mice were compared to vehicle-treated and untreated mice. Mean ± SEM. Two-way ANOVA. Tukey’s multiple comparisons test between groups. (**** p < 0.0001, *** p < 0.001). Untreated N = 5, vehicle treated N = 7, FTY720 treated N = 6. D At the end of the light damage experiments, mice were culled, and the eyes were enucleated to perform further histological investigations. Rodents eyes were fixed in 4% PFA, incubated in 30% sucrose for 1–2 days, embedded in OCT (embedding matrix), cryosectioned and stained with DAPI. IHC of untreated, vehicle or FTY70 treated Rho M39R/+ superior retina after light damage. The cryosections were stained with rhodospin-4D2 (in magenta) and anti-GFAP (in yellow). Scale bar = 20 μm. E The % area occupied by GFAP-positive signal relative to the ONL, OPL, and INL was measured after thresholding GFAP staining in untreated and treated animals. Analysis performed in Fiji. Mean ± SEM. Two-way ANOVA. Tukey’s multiple comparisons test between groups. (** p < 0.001, *** p < 0.001). N = 3.

Article Snippet: Mice in dim red light were injected intraperitoneally with vehicle (saline solution) or FTY720 (Bio-techne, Tocris, 1 mg/ml in saline solution).

Techniques: RNA Sequencing, Control, Injection, Saline, Incubation, Staining

Journal: Cell Death Discovery

Article Title: Preventing light-induced toxicity in a new mouse model of sector retinitis pigmentosa caused by Rhodopsin M39R variant

doi: 10.1038/s41420-025-02769-2

Figure Lengend Snippet: A Rho M39R/M39R KI mice were treated with FTY720 at 4 weeks of age. Schematic of the FTY20 treatment in Rho M39R/M39R KI mice. Mice were dark-adapted and injected with FTY720 (10 mg/kg) 30 min prior to the light damage assay. The ERG was performed once to induce a faster degeneration. The OCT was performed at both day 0 and day 2, after 48 h from the single ERG. B The ONL thickness was measured at day 2, after a single ERG. FTY720-treated Rho M39R/M39R KI mice were compared to vehicle-treated and untreated mice. Mean ± SEM. Two-way ANOVA. Tukey’s multiple comparisons test between groups. (**** p < 0.0001, *** p < 0.001). Untreated N = 6, vehicle and FTY720 treated N = 5. C IHC of untreated, vehicle or FTY70 treated Rho M39R/M39R superior retina after light damage. The cryosections were stained with rhodospin-4D2 (in magenta). Scale bar = 20 μm. D The number of photoreceptors in the ONL was measured at 200–400 μm from the optic nerve in the inferior and superior retina. The analysis was performed on images of the central retina acquired with a microscope EVOS FL auto 2. The area of 10–20 nuclei per retina was measured and divided to total area of the ONL to calculate the total number of nuclei. The number of photoreceptors in 100 μm per treated/untreated animal was plotted. Mean ± SEM. Two-way ANOVA. Tukey’s multiple comparisons test between groups. (* p < 0.05, ** p < 0.01). N = 3.

Article Snippet: Mice in dim red light were injected intraperitoneally with vehicle (saline solution) or FTY720 (Bio-techne, Tocris, 1 mg/ml in saline solution).

Techniques: Injection, Staining, Microscopy

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: Cells were incubated with FTY720 and FTY720 (S)-phosphate (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) or cycloheximide (Sigma, St. Louis, MO, USA) at 3 3 10/ml for 3 h. In some cases, neutrophils were preincubated for 30 min with necrostatin-1 or Z-IETD-FMK (both from Sigma); Q-VD-OPh (R&D Systems, Minneapolis, MN, USA); NSA (Tocris, Bristol, United Kingdom); geldanamycin, 17-DMAG, and radicicol (all from InvivoGen, San Diego, CA, USA); OA (Santa Cruz Biotechnology); or DPI (Sigma).

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

Journal: Frontiers in Immunology

Article Title: Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4 + Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

doi: 10.3389/fimmu.2017.01622

Figure Lengend Snippet: The expansion of the CD11c hi MHCII + CD4 + DC population in the bone marrow is mediated by S1P receptors and does not rely on splenic dendritic cells (DCs). (A) Mice were treated with the functional sphingosine 1-phosphate (S1P) receptor antagonist FTY720 or with the solvent as control before sham or cecal ligation and puncture (CLP) operation. After 36 h, the number of CD11c hi MHCII + CD4 + DC in the bone marrow was determined. The scatter plot shows the values of individual mice ( n = 5–9 mice per group) and the median/interquartile range as horizontal lines. Statistical differences were tested using the Kruskal–Wallis test followed by Dunn’s posttest. (B,C) CD11c hi MHCII + CD4 + DCs in the spleen. The gating strategy (B) was equivalent to the gating of CD4 + DCs in the bone marrow. Numbers indicate the percentage of gated cells or cells within the respective quadrant. Representative dot plots of one sham and one CLP mouse (36 h after surgery) are shown. (C) At different time points after sham or CLP operation total spleen cells were isolated and the number of CD11c hi MHCII + CD4 + DCs was quantified ( n = 4–21 per group). (D) Mice underwent splenectomy or laparotomy as control surgery 4 weeks before sham or CLP operation. After 36 h, the number of CD4 + DC in the bone marrow was quantified. The scatter plot shows the values of individual mice. Horizontal lines depict the median/interquartile range ( n = 7–11 mice per group). Statistical differences between sham and CLP were tested using the Mann–Whitney U -test. * p < 0.05; *** p < 0.001. DCs, dendritic cells.

Article Snippet: Where indicated, the functional S1P receptor antagonist FTY720 (2 mg/kg body weight; Fingolimod, LC Labs, Woburn, MA, USA) dissolved in 20% 2-hydroxypropyl-β-cyclodextrin (2-HPCD) was intravenously (i.v.) applied immediately before sham and CLP operation and 12 h thereafter.

Techniques: Functional Assay, Solvent, Control, Ligation, Isolation, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4 + Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

doi: 10.3389/fimmu.2017.01622

Figure Lengend Snippet: CD4 + T cells accumulate in parallel to CD4 + DCs in the bone marrow during sepsis. (A) Representative dot plots of bone marrow cells (BMC) from one sham and one cecal ligation and puncture (CLP) mouse 36 h after surgery. CD11c − CD4 + T lymphocytes were gated. (B,C) Mice were treated with the functional sphingosine 1-phosphate receptor antagonist FTY720 or with the solvent before sham or CLP operation. After 36 h, the number and frequency of CD4 + T cells in the bone marrow (B) and blood (C) , respectively, were determined. Data show the values of individual mice ( n = 5–9 mice per group) with median/interquartile range. (D) Analysis of the bone marrow of one representative naive wildtype (wt) and one Rag2 −/− mouse for the presence of CD4 + T cells. Numbers indicate the percentage of gated cells. (E) Wildtype and Rag2 −/− mice underwent sham or CLP operation. After 36 h, BMC were isolated and the frequency of CD11c hi MHCII + CD4 + DCs was determined according to the gating strategy shown in Figure B. Data indicate the values of individual mice ( n = 4–8 per group). Horizontal lines depict the median/interquartile range. Significant differences between sham and CLP were tested using the Mann–Whitney U -test. * p < 0.05; ** p < 0.01; *** p < 0.001. DCs, dendritic cells.

Article Snippet: Where indicated, the functional S1P receptor antagonist FTY720 (2 mg/kg body weight; Fingolimod, LC Labs, Woburn, MA, USA) dissolved in 20% 2-hydroxypropyl-β-cyclodextrin (2-HPCD) was intravenously (i.v.) applied immediately before sham and CLP operation and 12 h thereafter.

Techniques: Ligation, Functional Assay, Solvent, Isolation, MANN-WHITNEY

Journal: Rheumatology (Oxford, England)

Article Title: FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα–CPT1a pathway

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: FTY720 alleviated renal injury in EAV rats. ( A , B ) Representative images of glomeruli ( A ) and tubulointerstitium ( B ) of PAS staining for kidneys of rats (scale bar: 100 µm). ( C , D ) Representative images of glomerular ( C ) and tubulointerstitial ( D ) neutrophil infiltration in EAV rats (scale bar: 50 µm). ( E , F ) Amelioration of haematuria ( E ) and proteinuria ( F ) of EAV rats following FTY720 treatment. ( G , H ) Assessment of crescent formation ( G ) and TIN score ( H ) in EAV rats following FTY720 treatment. ( I , J ) Quantification of infiltrating neutrophils in glomeruli ( I ) and tubulointerstitium ( J ). Data are presented as means ( s . d .) from five rats per group. * P < 0.05, ** P < 0.01, *** P < 0.001; ns: not significant. DAPI: 4′,6-diamidino-2-phenylindole; EAV: experimental autoimmune vasculitis; HSA: human serum albumin; MPO: myeloperoxidase; PAS: periodic acid–Schiff; TIN: tubulointerstitial nephritis

Article Snippet: According to the product instruction, FTY720 (A8548, APExBIO, Houston, TX, USA) was dissolved in a mixture of 10% dimethyl sulfoxide, 40% PEG300 and 50% phosphate-buffered saline (PBS).

Techniques: Staining

Journal: Rheumatology (Oxford, England)

Article Title: FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα–CPT1a pathway

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: RNA sequencing analysis and gene enrichment analysis of renal cortex from rats. ( A , B ) Heat map ( A ) and scatter plot ( B ) presenting the distribution of DEGs between vehicle-treated and FTY720-treated groups. Fold change >2.0 or <0.5, P < 0.05. ( C ) A cluster profiler of the 10 most prominently enriched pathways identified in RNA sequencing and gene enrichment analysis between vehicle-treated and FTY720-treated groups. ( D , F ) Gene set enrichment analysis of FAO ( D ), PPAR signalling pathway ( E ) and respiratory burst pathway ( F ). FTY represents EAV rats treated with FTY720; AAV represents EAV rats treated with vehicle. AAV: ANCA-associated vasculitis; DEG: differentially expressed gene; FAO: fatty acid oxidation; FC: fold change; FDR: false discovery rate; NES: normalized enrichment score; PPAR: peroxisome proliferator activated receptor

Article Snippet: According to the product instruction, FTY720 (A8548, APExBIO, Houston, TX, USA) was dissolved in a mixture of 10% dimethyl sulfoxide, 40% PEG300 and 50% phosphate-buffered saline (PBS).

Techniques: RNA Sequencing

Journal: Rheumatology (Oxford, England)

Article Title: FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα–CPT1a pathway

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: FTY720 inhibited ANCA-induced neutrophil activation while upregulating FAO. ( A ) Identification of increased neutrophil marker CD11b of differentiated HL-60 cells. ( B ) Inhibitory effect of FTY720 on respiratory burst of differentiated HL-60 cells stimulated with serum from AAV patients. ( C ) Inhibitory effect of FTY720 on respiratory burst of isolated human neutrophils activated by MPO-ANCA positive IgG. ( D ) Gene expression of the top six upregulated genes in FAO in RNA sequencing was measured in differentiated HL-60 cells treated with the 10% serum from AAV patients and FTY720 or vehicle. ( E ) mRNA level of PPARα and protein expression of CPT1a and PPARα in differentiated HL-60 cells treated with the 10% serum from AAV patients and FTY720 or vehicle. Data are shown as means ( s . d .). * P < 0.05, ** P < 0.01, *** P < 0.001; ns: not significant. AAV: ANCA-associated vasculitis; ABCD3: ATP binding cassette subfamily D member 3; CPT1a: carnitine palmitoyltransferase 1A; ECHDC2: enoyl-CoA hydratase domain containing 2; FAO: fatty acid oxidation; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HSD17b4: 17β-hydroxysteroid dehydrogenase 4; MFI: mean fluorescence intensity; PPARα: peroxisome proliferator activated receptor α; ROS: reactive oxygen species; SESN2: sestrin 2; SLC27A2: solute carrier family 27 member 2

Article Snippet: According to the product instruction, FTY720 (A8548, APExBIO, Houston, TX, USA) was dissolved in a mixture of 10% dimethyl sulfoxide, 40% PEG300 and 50% phosphate-buffered saline (PBS).

Techniques: Activation Assay, Marker, Isolation, Gene Expression, RNA Sequencing, Expressing, Binding Assay, Fluorescence

Journal: Rheumatology (Oxford, England)

Article Title: FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα–CPT1a pathway

doi: 10.1093/rheumatology/keae320

Figure Lengend Snippet: FTY720 exerted an inhibitory effect on ANCA-induced neutrophil activation via the PPARα–CPT1a pathway. ( A ) Determination of CPT1a-knockdown efficiency by qPCR and western blot. ( B , C ) The inhibitory effect of FTY720 on respiratory burst in differentiated HL-60 cells upon stimulation with 10% serum from AAV patients was reversed by CPT1 antagonist etomoxir ( B ) and CPT1a knockdown ( C ). ( D , E ) The effect of GW6471 (a PPARα antagonist) on the inhibition of FTY720 on respiratory burst ( D ) and NET formation ( E ). ( F , G ) The effect of combination of GW7647 (a PPARα agonist) and etomoxir (a CPT1 inhibitor) on the inhibition of FTY720 on respiratory burst ( F ) and NET formation ( G ). ( H ) Representative images of NET formation with or without FTY720, etomoxir, GW6471 (scale bar: 50 µm). Data are shown as means ( s . d .). * P < 0.05, ** P < 0.01, *** P < 0.001; ns: not significant. AAV: ANCA-associated vasculitis; CPT1a: carnitine palmitoyltransferase 1A; Eto: etomoxir; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; MFI: mean fluorescence intensity; PPARα: peroxisome proliferator activated receptor α; ROS: reactive oxygen species

Article Snippet: According to the product instruction, FTY720 (A8548, APExBIO, Houston, TX, USA) was dissolved in a mixture of 10% dimethyl sulfoxide, 40% PEG300 and 50% phosphate-buffered saline (PBS).

Techniques: Activation Assay, Knockdown, Western Blot, Inhibition, Fluorescence