Fig 1: LIFR mediates the beneficial effects of CD8+ TRLs after stroke.(A) Transwell coculture system of brain slices collected 1 day after stroke and CD8+ TRLs from healthy spleens. RT-PCR analysis of Lifr, Tgfa, and Il10 24 hours after coculture. Kruskal-Wallis test and post hoc Dunn’s test. n = 6–16/group. (B) Anti-LIF antibody (60 ng/mL) was added to the CD8+ TRL–brain slice coculture system (as in A). The expression of Tgfa and Il10 was measured by RT-PCR 24 hours after coculture. n = 4–6/group. One-way ANOVA and post hoc Bonferroni’s test. CL, contralateral brain; IP, ipsilateral brain. (C–G) Spleen-derived CD8+ TRLs were pretreated with LIF (100 ng/mL), LIFR inhibitor (EC359, 100 nM), or PBS for 1 hour and then injected (i.v., 1 × 106 cells) into recipient mice 2 hours after tMCAO. (D and E) LIF treatment enhanced ETGF and IL-10 expression in CD8+ TRLs. n = 3/group. Two-tailed Student’s t test. (F) Quantification of MAP2 staining and (G) Garcia score. n = 5–6/group. One-way ANOVA and post hoc Bonferroni’s test. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 2: LIFR and ETGF are upregulated in CD8+ TRLs after stroke.(A) CD8+ TRLs from ischemic brain 3 days after tMCAO and from blood after sham operation were subjected to quantitative PCR array. The gene expression was normalized to the corresponding blood level. Heatmap showing the log2(fold change) for genes with >2-fold changes. n = 3/group. *FDR < 0.2, **FDR < 0.1 for genes upregulated (red) or downregulated (blue) in brain-infiltrating CD8+ TRLs. (B) Western blot analysis of LIFR and ETGF expression in the brain lysates collected from sham mice and 1, 3, 5, and 7 days after stroke. n = 4/group. One-way ANOVA and post hoc Dunnett’s test. (C) MFI of LIFR in CD8+CD122+ TRLs in the blood (Bl), spleen (Sp), and ipsilateral brains (Br) 3 days after tMCAO or sham operation. One-way ANOVA and post hoc Dunnett’s test. Iso, isotype control. (D) Immunostaining of LIF 3 days after stroke. Images are representative of 4 animals in each group. (E) Expression of LIF was assessed by ELISA 3 days after tMCAO. n = 6/group. One-way ANOVA and post hoc Bonferroni’s test. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 3: Control and LtfCre ESR1flox/flox mice with branched glands (blue) display threshold levels of LIF production permitting proper embryo (purple) axis alignment and V-shaped implantation chamber formation (pink cells) allowing embryo development to term. In contrast, the branchless glands in PgrCre ESR1flox/flox and Pax2Cre ESR1flox/flox mice compromise LIF secretion, resulting in implantation failure. GD: gestational day; LIF: leukemia inhibitory factor; ESR1: estrogen receptor 1; Ltf: lactoferrin; Pgr: progesterone; Pax2: paired box gene 2.
Fig 4: LIF initiates implantation in FOXA2-deficient mice. a In situ localization of Hbegf mRNA in the uterus of control and FOXA2-deficient mice without LIF repletion on GD 4 at 2200 hours. Uterine sections were counterstained with hematoxylin after chromogenic detection of Hbegf mRNA (red). Top panel—Scale bar: 500 μm; Bottom panel—Scale bars: 25 μm. b Gross morphology of uteri on GD 5 at 0800 hours. FOXA2-deficient mice received intraperitoneal (i.p.) injections of saline or recombinant mouse LIF on GD 4. Implantation sites accumulate Evans Blue Dye. White arrowheads point to individual implantation sites. Scale bar: 1 cm. c In situ localization of Hbegf mRNA in the uterus on GD 5 at 0800 hours. FOXA2-deficient mice received intraperitoneal (i.p.) injections of saline or recombinant mouse LIF on GD 4. Scale bar: 50 μm. AM: antimesometrial, M: mesometrial, Em: embryo, LE: luminal epithelium, S: stroma. All images are representative of three independent experiments
Fig 5: Stromal cell decidualization and epithelial remodeling in LIF-replaced FOXA2-deficient mice. Control, gland-containing LtfiCre/+Foxa2f/f and glandless PgrCre/+Foxa2f/f mice received i.p. injections of recombinant mouse LIF on GD 4 and were evaluated on GD 5 at 0800 hours. a Immunofluorescence analysis of PTGS2 and CK8 in implantation sites. PTGS2 is present in the stroma around the implanting embryo on the antimesometrial side of the uterus. Scale bar: 100 μm. b Immunofluorescence analysis of CDH1 in implantation sites. Scale bar: 100 μm. c Immunofluorescence analysis of CLDN1 in implantation sites. Scale bar: 100 μm. AM: antimesometrial, M: mesometrial, Em: embryo, LE: luminal epithelium, S: stroma. All images are representative of three independent experiments
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