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: 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
Fig 4: Pre-pubertal, unbranched glands fail to produce leukemia inhibitory factor even when estrogen receptor 1 signaling is intact. (A) Uterine sections of P21 control mice injected with either oil, E2, or E2 + P4 and stained for Hoechst (nuclei, white), FOXA2 (gland marker, green), ESR1 (a–c, red), and Lif mRNA (d–f, red). Scale bar, 40 μm (a–c). Scale bar, 80 μm (d–f). n = 9 regions each from three mice per treatment. Quantitative analysis of (B) the amount of Lif normalized for gland volume and (C) the amount of Lif normalized for lumen volume. n = 9 regions each from three mice per treatment. (B and C) Data analyzed using Mann–Whitney test. (*) = P < 0.05. Yellow arrowheads point towards the luminal epithelial cells and white arrows points towards the glandular epithelial cells (D) Representative 3D reconstructions of P21 control uterine glands with various treatments. n = 3 mice per treatment group. Scale bar, 100 μm. Quantitative analyses of (E) average gland length measurements (each dot represents one mouse) and (F) percentage of glands with 0, 1–3, and >3 branches in P21 control mice with various treatments. Hormonal treatment does not affect gland length or gland branching. 85–450 glands analyzed per mouse. (E) Data analyzed using Mann–Whitney test. (*) = P < 0.05. Two-proportion Z-test determined that the differences in percentage of gland branches between oil, E2, and E2 + P4 treatment is not statistically significant. FOXA2, forkhead box A2; ESR1, estrogen receptor 1; LIF, leukemia inhibitory factor; P, postnatal day; GD, gestational day; E2, estrogen; P4, progesterone.
Fig 5: Epithelial estrogen receptor 1 deletion mice display defects in leukemia inhibitory factor expression. (A) Uterine sections of control, LER, and XER mice at GD3 1200 h and GD3 1800 h with staining for Hoechst (nuclei, white), FOXA2 (gland marker, green), and Lif mRNA (red). Lif expression in LER glands is high in a subset of cells (b–b″, g–g″) and lower in others (c–c″, g–g″). sLif expression increases from GD3 1200 h to GD3 1800 h in LER glands while XER glands express undetectable levels of Lif at both time points. Scale bar, 100 μm. (B) Quantitative analysis of the amount of Lif per gland volume (normalized units) in control, XER, and LER mice at GD3 1200 h and GD3 1800 h. n = 9 regions each from three mice per genotype per stage were analyzed. Data in (B) analyzed using Kruskal–Wallis test with Dunn’s multiple comparisons. (*) = P < 0.05. (C) Uterine horn of XER mouse intraluminally injected with 1 μg recombinant Lif into the right horn on GD3 1200 h and dissected on GD4 1200 h following blue dye injection. Asterisks indicate implantation rescue sites, and the total number of embryos found in each horn is shown. ESR1, estrogen receptor 1; LER, LtfCre ESR1flox/flox (adult uterine epithelial deletion of ESR1 using lactoferrin promoter driven CRE expression); XER, Pax2Cre ESR1flox/flox (embryonic uterine epithelial deletion of ESR1 using paired box 2 (PAX2) promoter-driven CRE expression); FOXA2: forkhead box A2; LIF, leukemia inhibitory factor; GD, gestational day; em, embryo.
Supplier Page from BioLegend for Recombinant Mouse LIF (carrier-free)