Fig 1: Signalling kinetics elicited by S1P or FTY720-P.a HEK293 cells expressing S1PR1 were S1P-starved for 24 h, with or without 300 ng/mL pertussis toxin (PTX) for 18 h and with 0.1% DMSO alone or with 50 µM CCG215022 (CCG) for 30 min prior of being stimulated with 0.1 µM S1P or FTY720-P for the indicated times. pERK1/2 was measured in the cell extracts by ELISA and expressed as relative units; n = 3 independent experiments; hereafter, ELISA time points are given as mean values ± SEM. b Protein extracts from cells treated as in a for the indicated times were assayed by immunoblot for S1PR1, ERK1/2 and pERK1/2.
Fig 2: The decreased level of S1PR1 and increased level of miR-223-3p transcripts in CD4+ T cells from SLE patients. (A) mRNA expression of S1PR1 and miR-223-3p in CD4+ T cells isolated from healthy (n = 6) and the patients with SLE (n = 15). Data are presented as mean ± SEM. *p < 0.05, ns, not significant, by Wilcoxon signed-rank test. (B, C) The miR-223-3p and S1PR1 transcript level in CD4+ T cells isolated from the SLE patients with organ involvement and no involvement. SLE patients with skin involvement (n = 11) and without involvement (n = 4), with lung involvement (n = 2) and without involvement (n = 13), with central nervous system involvement (n = 4) and without involvement (n = 11), with kidney involvement (n = 6) and without involvement (n = 9), with joints involvement (n = 10) and no involvement (n = 5). Data are presented as mean ± SEM. *p < 0.05, ns, not significant, by Student’s t-test.
Fig 3: Top 20 differential expression levels of miRNAs and mRNAs in CD4+T cells isolated from MRL/lpr lupus-prone (MRL/lpr) vs C57BL/6 (B6) mice by microarray analysis. (A) Fold change (ratio between MRL/lpr/B6) in miRNA expression MRL/lpr vs B6. We focused on mmu-miR-223-3p which was indicated by arrow. (B) Candidate target genes for upregulated mmu-miR-223-3p were identified using the commonly used prediction algorithm, miRDB. These targets and our mRNA expression profiles were integrated. Fold change (ratio between MRL/lpr/B6) in mRNA expression MRL/lpr vs B6. We focused on S1pr1 which was indicated by arrow. (C) The expression level of miR-223-3p was evaluated by TaqMan quantitative PCR in MRL/lpr mice and B6/lpr lupus-prone (B6/lpr) mice compared with B6 mice (n = 5 per group, 16-week-old female). (D) The expression of S1pr1 in CD4+T cells of mice spleen was evaluated by TaqMan quantitative PCR (n =5 per group, 16-week-old female). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, by Student’s t-test.
Fig 4: Glomerulonephritis with infiltration of CD4+S1PR1+ T cells in in B6-Mir223-/-Faslpr/lpr (A) The image of CD4+ cells, CD4+S1PR1+ cells, CD8+ cells and CD8+S1PR1+ cells in glomerular lesion in B6-Mir223-/-Faslpr/lpr compared to B6-Mir223+/+Faslpr/lpr mice (n = 5 per group). Images are representative of five mice per group. Bars = 50 µm. (B) The number of CD4+ cells, CD4+S1PR1+ cells, CD8+ cells and CD8+S1PR1+ cells in glomerulus or tubular lesion in B6-Mir223-/-Faslpr/lpr compared to B6-Mir223+/+Faslpr/lpr mice (n = 5 per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ns, not significant, by Student’s t-test in (B).
Fig 5: The proportion of CD3+ T cells, CD3+CD4-CD8- T cells, CD19+ B cells, CD19-CD138+ cells (Plasma cells), CD3+S1PR1+ T cells and CD3+CD4+S1PR1+ T cells in spleen and early apoptotic cells in CD4+ and CD8+ T cells in lymph nodes were significantly increased in B6-Mir223-/-Faslpr/lpr mice. (A) Various cellular subsets in lymph nodes and spleen. CD3+CD4+ (CD4 T cells), CD3+CD8+ (CD8 T cells), CD3+ CD4-CD8- (Double negative (DN) T cells), CD19+ (B cells), CD19-CD138+ (Plasma cells), CD19+CD138+ (Plasmablasts), CD4+CD44+CD62L+ (Memory T cells), CD4+CD44+CD62L- (Effector T cells), CD69+CD4+ cells, S1PR1+CD3+ cells, S1PR1+CD4+ cells. Distribution of subsets in total cells isolated from whole cervical lymph nodes and spleen were indicated in B6-Mir223-/-Faslpr/lpr (n=7) and B6-Mir223+/+Faslpr/lpr (n = 8) mice. Absolute number of total splenocyte and lymph nodes cell had no difference between two genotypes (Supplementary Figure 2E). (B) Apoptotic cells in lymph nodes and spleen. Annexin V+7-AAD- (Early apoptosis cells), Annexin V+7-AAD+ (Late apoptotic cells). Distribution of subsets in CD3+CD4+ T cells or CD3+CD8+ T cells isolated from whole cervical lymph nodes and spleen were indicated in B6-Mir223-/-Faslpr/lpr (n = 3) and B6-Mir223+/+Faslpr/lpr (n = 3) mice. *p < 0.05, **p < 0.01, ns, not significant, by Student’s t-test in (A), by Wilcoxon signed-rank test in (B).
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