Fig 1: LIF promotes BCE cell growth via the JAK-STAT3 pathway AThe JAK inhibitor baricitinib (Ba) blocked LIF-induced STAT3 phosphorylation. BCE cells were preincubated with DMSO, baricitinib (2 µM), cobimetinib (Co) (150 nM), or BEZ235 (BE) (5 nM) for 1 h and were then treated with vehicle or LIF (10 ng/ml, Sigma) for 15 min. Ctrl, no preincubation with inhibitors.BBaricitinib suppressed LIF-induced BCE cell growth. BCE cells were preincubated with DMSO, baricitinib, cobimetinib, or BEZ235 for 1 h and then treated with vehicle, LIF (10 ng/ml), or VEGF (10 ng/ml). Cell proliferation was analyzed after 6 days, n = 3.C, DSTAT3 knockdown in BCE cells. BCE cells were transfected with siNegative and siRNAs targeting STAT3. qRT-PCR was performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and are presented in C. In D, cells transfected with siRNAs were treated with LIF (10 ng/ml) or vehicle for 15 min. Whole-cell lysates were subjected to Western blotting with the indicated antibodies.ELIF-induced BCE cell growth was abolished by STAT3 knockdown. BCE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) or vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading at 590 nm for each vehicle group was set as 1, n = 3. siNegative, negative control siRNA not targeting any known genes. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. Source data are available online for this figure.
Fig 2: LIF inhibits BAE cell growth through the JAK-STAT3 pathway ARecombinant human LIF inhibited growth of BAE cells in a dose-dependent manner. BAE cells were cultured in the presence of vehicle and indicated concentrations of recombinant human LIF (rhLIF). Cell proliferation was analyzed after 6 days, n = 3.BJAK inhibitor baricitinib blocked activation of STAT3 by LIF. BAE cells preincubated with DMSO and inhibitors for 1 h were treated with vehicle and LIF (10 ng/ml) for 15 min. Whole-cell lysates were subjected to Western blotting with indicated antibodies. Ctrl, no preincubation with inhibitors; Ba, baricitinib (2 µM); Co, cobimetinib (150 nM); BE, BEZ235 (5 nM).CThe JAK inhibitor baricitinib reversed LIF-induced BAE growth inhibition. BAE cells preincubated with inhibitors for 1 h were treated with vehicle and LIF (10 ng/ml, Sigma). Cell proliferation was analyzed after 6 days using alamar blue, n = 3.D, EKnockdown of STAT3 in BAE cells. BAE cells were transfected with siRNAs targeting STAT3. qRT-PCR was performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and shown in D. In E, cells transfected with siRNAs were treated with LIF (10 ng/ml, Sigma) and vehicle for 15 min. Whole-cell lysates were subjected to Western blotting with indicated antibodies.FLIF-induced BAE cell growth inhibition was abolished by knockdown of STAT3. BAE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) and vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading for each vehicle group was set as 1, n = 3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two-way ANOVA was used as statistical test. Source data are available online for this figure.
Fig 3: LIF promotes angiogenesis in in vivo models A, BIntravitreal injection of LIF increases blood vessel density in the mouse eyes. Adult mice were intravitreally injected with VEGF (10 ng) or LIF (10–100 ng, Sigma). Seven days after injection, PFA-fixed choroid–sclera complexes and retina were subjected to CD31 IF. Representative images of CD31-positive vessels are shown in A. Scale bar = 100 µm. Vascular density, determined with ImageJ software, is shown in B), n = 5–8.C, DOCT-A imaging of LIF-treated mouse retina. Adult mice were intravitreally injected with 1 µl of LIF (50 ng, Sigma) or vehicle solution (PBS). Retinal OCT-A images were obtained 7 days after the injection and representative images are shown. Blood vessel density was determined as percentage of vessel-covered area/total area surface using ImageJ software and shown in D), n = 7–8.ESTAT3 was phosphorylated in retinal ECs after LIF treatment. Fifty nanogram of our in-house LIF with low endotoxin levels was injected. Phospho-Stat3 was examined on cryosection of retina after 2 h by IHC. Magnification in the dashed box is shown in the lower panel. n = 5. Scale bar = 100 µm.FFive-day-old neonatal mice were intravitreally injected with LIF (50 ng) or vehicle solution (PBS). After 3 days, retinas were subjected to IF staining with Dyight-488-labeled lectin. Representative images for similar ocular loci and quantification of lectin-labeled area using ImageJ software were shown, n = 4. Scale bar = 100 µm. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. Source data are available online for this figure.
Fig 4: Effects of LIF and other IL-6 family members in mouse ocular models IF enhanced laser-induced CNV. Intravitreal administration of LIF (10 ng and 100 ng, Sigma) and OSM (10 ng) after laser-induced CNV. PBS was used as vehicle control. OCT-A imaging and immunostaining of choroid flat mounts were carried out 10 days after laser induction. Quantification of CNV area is shown. Dashed line circles indicate the CNV in OCT-A images. n = 5. Dot plot shows all data points from three independent experiments. Scale bar = 100 µm.Sodium iodate was administered to induce retinal and CC damage in mice. After sodium iodate injection, the indicated amounts of LIF, CT-1, or OSM were injected in the eyes. Choroid capillaries were imaged by OCT-A system, n = 5. The avascular area in CC is indicated in yellow.H & E staining of eyes treated with different IL-6 proteins following sodium iodate injection (n = 5). Representative images are shown. Scale bar = 100 µm.Avascular area in choroid in sodium iodate model was determined and quantified using Image J.Thickness of retina in H&E staining was quantified and bar graph was shown using ImageJ. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. Source data are available online for this figure.
Fig 5: Expression of LIF and LIFR in ECs Q-PCR (Taqman) analysis of LIF expression in BCE cells and bovine pericytes (bPericyte).Bovine LIF ELISA of BCE cells and bovine pericyte. Cells were cultured for 48 h before harvesting the medium.LIFR expression in BCE cells and bovine pericyte by qPCR (Taqman).Image of FACS selection of CD13+/CD45- and CD31+/CD45- cells.Immunostaining for CD31, CD13, and DAPI in isolated primary mouse cells before and after FACS selection.Mouse LIF expression in FACS-selected cells.Human choroidal EC (HCEC) and mouse retinal EC (MREC) were treated with LIF 10 ng and LIF 100 ng for 30 min. (PBS was used as control). STAT3 phosphorylation was detected by Western blot.H&E staining of LIFR and CD31 in mouse eye sections. The magnified images of the dashed line boxes, a and b, are shown on the right panel. Box a shows LIFR staining in retina and box b showed LIFR staining in RPE/choroid layer.Whole-mount staining of LIFR and CD31 in mouse retina. The fluorescent staining for CD31 (green) and LIFR (red) is shown. White arrows indicate the co-staining of CD31 and LIFR on retinal ECs. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.
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