Fig 2: Identification of LIF as the EC mitogen in LN-229-conditioned medium (CM) LN-229 CM stimulated growth of BCE cells, n = 3.VEGF neutralizing antibody (B20) failed to suppress BCE cell growth induced by LN-229 CM, n = 3.Reverse-phase fractions of LN-229 CM induced BCE cell growth. BCE cells were incubated with fractions (2 µl/well) as indicated in the figure, n = 3.Candidate proteins generated from mass-spectrometry analysis of LN-229 CM reverse-phase fractions. Candidates were identified by excluding intracellular proteins and proteins showing higher abundance in inactive fractions compared to those in mitogenic factions. Proteins were ranked for relative abundance as described in Materials and Methods.The anti-LIF neutralizing antibody abolished BCE cell growth induced by reverse-phase fractions, n = 3.Recombinant human LIF proteins stimulated growth of BCE cells in a dose-dependent manner. BCE cells were cultured in the presence of vehicle, VEGF (10 ng/ml), and the indicated concentrations of recombinant human LIF (rhLIF, Sigma), n = 3.LIF and VEGF synergistically stimulated BCE cell growth. Cell proliferation was analyzed after 6 days using alamar blue as described in Materials and Methods, n = 3. 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. ns, not statistically significant. Source data are available online for this figure.

Fig 3: 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 4: 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.

Fig 5: 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.