Journal: The Journal of Neuroscience

Article Title: Neuroprotective and Axon Growth-Promoting Effects following Inflammatory Stimulation on Mature Retinal Ganglion Cells in Mice Depend on Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor

doi: 10.1523/JNEUROSCI.2770-09.2009

Figure Lengend Snippet: Changes in the expression of CNTF and LIF, as well as JAK/STAT3 pathway activation, in the murine retina after ONC and LI. A, Immunohistochemical staining of an untreated control retina (con), a retina 5 d after optic nerve crush (onc), and onc + lens injury (li) from wild-type mice as indicated using an anti-CNTF (red) and an anti-GFAP (green) antibody. Scale bar, 50 μm. GCL, ganglion cell layer; INL, inner nuclear layer. B, Western blot analysis of retinal lysates from wild-type (wt) and CNTF-deficient (cntf−/−) mice 5 d after onc, onc + li, or no previous treatment (con) using specific antibodies against GFAP, GAP43, LIF, phospho-STAT3 (pSTAT3), or CNTF. Tubulin served as a loading control. C, Immunohistochemical staining of astrocytes of an untreated control retina, a retina 5 d after onc, and after onc + li of wt and cntf−/− mice using an anti-LIF (R&D) (red) and anti-GFAP antibody (green). Scale bar, 25 μm. D, Quantitative real-time PCR. LIF expression levels were quantified relative to GAPDH in wt and cntf−/− mice 5 d after onc, onc + li, or untreated animals (−). *p < 0.05; ***p < 0.001. E, Western blot analysis of retinal lysates from CNTF/LIF double knock-out (cntf−/−/lif−/−) mice 5 d after onc, onc + li, or no previous treatment (−) using specific antibodies against GAP43, GFAP, and pSTAT3. Tubulin expression verified that the same amount of retinal protein was loaded per lane.

Article Snippet: They were then processed for immunostaining with either an antiserum against rat phospho-STAT3 (Tyr705) (Cell Signaling Technology) (1:5000), a monoclonal antibody against rat β-actin (Sigma) (1:7500), a monoclonal antibody against GFAP (Santa Cruz Biotechnology) (1:5000), a βIII-tubulin antibody (TUJ-1) (Babco) (1:1000) and an antibody against LIF (Santa Cruz Biotechnology) (1:6000), a polyclonal antibody against rat CNTF (Serotec) (1:5000), or a custom made antibody against GAP43 (Invitrogen) (1:1000) at 4°C overnight.

Techniques: Expressing, Activation Assay, Immunohistochemical staining, Staining, Control, Western Blot, Real-time Polymerase Chain Reaction, Knock-Out

Journal: The Journal of Neuroscience

Article Title: Neuroprotective and Axon Growth-Promoting Effects following Inflammatory Stimulation on Mature Retinal Ganglion Cells in Mice Depend on Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor

doi: 10.1523/JNEUROSCI.2770-09.2009

Figure Lengend Snippet: Regenerative state of RGCs 5 d after ONC and ONC + LI in mature wild-type and CNTF-deficient mice. A, Dissociated retinal cell cultures immunostained with an antibody against βIII-tubulin, showing spontaneously regenerating RGCs after 24 h in culture. Wild-type (wt) and CNTF-deficient (cntf−/−) mice had received ONC (onc), ONC + LI (onc + li), or no treatment (con) 5 d previously. Scale bar, 50 μm. B, Quantitation of neurite outgrowth of groups in A and of CNTF/LIF double knock-out (cntf−/−/lif−/−) mice 5 d after ONC + LI, indicated as average neurite length per RGC. C, Quantitation of RGCs per well after 24 h in culture, demonstrating no significant differences between groups. Significance between groups: ***p < 0.001; ns., nonsignificant.

Article Snippet: They were then processed for immunostaining with either an antiserum against rat phospho-STAT3 (Tyr705) (Cell Signaling Technology) (1:5000), a monoclonal antibody against rat β-actin (Sigma) (1:7500), a monoclonal antibody against GFAP (Santa Cruz Biotechnology) (1:5000), a βIII-tubulin antibody (TUJ-1) (Babco) (1:1000) and an antibody against LIF (Santa Cruz Biotechnology) (1:6000), a polyclonal antibody against rat CNTF (Serotec) (1:5000), or a custom made antibody against GAP43 (Invitrogen) (1:1000) at 4°C overnight.

Techniques: Quantitation Assay, Knock-Out

Journal: The Journal of Neuroscience

Article Title: Neuroprotective and Axon Growth-Promoting Effects following Inflammatory Stimulation on Mature Retinal Ganglion Cells in Mice Depend on Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor

doi: 10.1523/JNEUROSCI.2770-09.2009

Figure Lengend Snippet: Axon regeneration and survival of RGCs in vivo after LI in wild-type, CNTF-deficient and CNTF/LIF double knock-out mice. A, Longitudinal sections through the optic nerve showing GAP43-positive axons distal to the injury site (asterisk) in wild-type (wt), CNTF-deficient (cntf −/−) and CNTF/LIF double knock-out (cntf −/−/lif −/−) mice 2 weeks after ONC alone (onc) or ONC + LI (onc + li). Scale bar, 100 μm. B, Quantification of axon regeneration (number of axons growing 0.25, 0.5, and 1 mm beyond the injury site per optic nerve) 2 weeks after ONC alone or ONC + LI. C, Quantification of surviving RGCs (βIII-tubulin-positive RGCs per retinal cross-section) 2 weeks after ONC alone, ONC + LI, or untreated retinas (−). *p < 0.05; *** p < 0.001.

Article Snippet: They were then processed for immunostaining with either an antiserum against rat phospho-STAT3 (Tyr705) (Cell Signaling Technology) (1:5000), a monoclonal antibody against rat β-actin (Sigma) (1:7500), a monoclonal antibody against GFAP (Santa Cruz Biotechnology) (1:5000), a βIII-tubulin antibody (TUJ-1) (Babco) (1:1000) and an antibody against LIF (Santa Cruz Biotechnology) (1:6000), a polyclonal antibody against rat CNTF (Serotec) (1:5000), or a custom made antibody against GAP43 (Invitrogen) (1:1000) at 4°C overnight.

Techniques: In Vivo, Knock-Out

Journal: The Journal of Neuroscience

Article Title: Neuroprotective and Axon Growth-Promoting Effects following Inflammatory Stimulation on Mature Retinal Ganglion Cells in Mice Depend on Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor

doi: 10.1523/JNEUROSCI.2770-09.2009

Figure Lengend Snippet: Neurite growth-promoting effects of LIF and CNTF on mature RGCs in culture. A, Quantitation of neurite outgrowth of RGCs in the presence of increasing concentrations of LIF (as indicated), CNTF and an anti-LIF-antibody (α-LIF) for 3 d. Values were normalized to untreated controls. Treatment effects compared with the group treated with vehicle only. *p < 0.05; ***p < 0.001. B, Quantitation of RGCs per well after 3 d in culture, demonstrating no significant differences between groups.

Article Snippet: They were then processed for immunostaining with either an antiserum against rat phospho-STAT3 (Tyr705) (Cell Signaling Technology) (1:5000), a monoclonal antibody against rat β-actin (Sigma) (1:7500), a monoclonal antibody against GFAP (Santa Cruz Biotechnology) (1:5000), a βIII-tubulin antibody (TUJ-1) (Babco) (1:1000) and an antibody against LIF (Santa Cruz Biotechnology) (1:6000), a polyclonal antibody against rat CNTF (Serotec) (1:5000), or a custom made antibody against GAP43 (Invitrogen) (1:1000) at 4°C overnight.

Techniques: Quantitation Assay

Journal: Cell Death & Disease

Article Title: Leukemia inhibitory factor drives glucose metabolic reprogramming to promote breast tumorigenesis

doi: 10.1038/s41419-022-04820-x

Figure Lengend Snippet: A Ectopic LIF expression (left panel) or treatment with rhLIF (right) enhanced lactate production of different breast cancer cell lines analyzed by measuring lactate levels in the medium using a lactate assay kit. B LIF neutralization antibody (LIF neu-ab) blocked the promoting effect of LIF on lactate production in MCF7, MDA-MB 231 and T47D cells. C Knockdown of endogenous LIF decreased lactate production in MDA-MB 231 cells. D , E Ectopic LIF expression enhanced glycolysis rates ( D ), whereas knockdown of endogenous LIF decreased glycolysis rates ( E ) in different breast cancer cell lines as calculated by ECAR measured by using a Seahorse analyzer. F The diagram of glycolysis. G – I The fold change of inter-metabolites in glycolysis resulted from ectopic LIF expression in MCF7 and MDA-MB 231 cells ( G ), rhLIF treatment in MCF7 cells ( H ) or LIF knockdown in MDA-MB 231 cells ( I ) as determined by LC/MS metabolomics analysis. J The fold change of inter-metabolites in glycolysis induced by ectopic LIF expression in MCF7 xenograft tumors determined by LC/MS metabolomics analysis. Data are presented as mean ± SD ( n = 3/group). * p < 0.05; ** p < 0.01; *** p < 0.001; unpaired Student’s t -test.

Article Snippet: LIF neutralization antibody (AF-250-NA) was purchased from R&D Systems.

Techniques: Expressing, Lactate Assay, Neutralization, Knockdown, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Death & Disease

Article Title: Leukemia inhibitory factor drives glucose metabolic reprogramming to promote breast tumorigenesis

doi: 10.1038/s41419-022-04820-x

Figure Lengend Snippet: A Glut1 mRNA levels were detected by quantitative real-time PCR (qPCR) in MCF7, MDA-MB 231, and T47D cells with or without ectopic LIF expression. B , C Ectopic LIF expression ( B ) or rhLIF treatment (100 ng/ml for 12 h) ( C ) promoted endogenous Glut1 PM translocation in MCF7, MDA-MB 231, and T47D cells as determined by Western-blot assays. D Knockdown of LIF decreased endogenous Glut1 PM translocation in MDA-MB 231 cells. E Ectopic LIF expression increased the PM translocation of ectopically expressed Myc-Glut1 in MCF7, MDA-MB 231 and T47D cells as determined by Western-blot assays. F LIF neutralization antibody (LIF neu-ab) largely abolished exogenous Glut1 PM translocation promoted by LIF. G The rhLIF treatment promoted exogenous Glut1 PM translocation in cells. H Knockdown of LIF decreased Myc-Glut1 PM translocation in MDA-MB 231 cells. I Ectopic LIF expression promoted Myc-Glut1 PM translocation (left panels) while knockdown of LIF decreased Myc-Glut1 PM translocation (right panels) in MDA-MB 231 cells as determined by IF staining assays. Scale bar, 10 μm. J Ectopic LIF expression promoted the PM translocation of Myc-Glut1 in MCF7, MDA-MB 231, and T47D cells as determined by flow cytometry assays. Left panels: representative images of flow cytometry analysis. Right panels: quantifications of relative fluorescence intensity of Myc-Glut1 on the cell membrane normalized with total Myc-Glut1 fluorescence intensity in cells. In A , J data are presented as mean ± SD. n = 3/group. * p < 0.05; ** p < 0.01; NS: non-significant; unpaired Student’s t -test. Uncropped Wes t ern-blot images are shown in Supplementary Fig .

Article Snippet: LIF neutralization antibody (AF-250-NA) was purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Translocation Assay, Western Blot, Knockdown, Neutralization, Staining, Flow Cytometry, Fluorescence, Membrane

Journal: Development (Cambridge, England)

Article Title: Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

doi: 10.1242/dev.134023

Figure Lengend Snippet: Ectopic IL6 family ligands result in accelerated AT2 differentiation via STAT3 activation. (A) Schematic and section of E15.5 slice culture resulting in differentiation of mature saccules with AT1 and AT2 cells in the presence of Dx. Green, pro-SFTPC; red, PDPN. (B) Schematic of IL6 and LIF experiments. Slices from individual lungs were split between two conditions for internal controls. (C,D) Sections from control, IL6- and LIF-exposed wild-type lungs. (C) Red, pSTAT3; white, E-CAD (epithelium). (D) Red, LAMP3 (differentiating AT2 cells); green LPCAT1 (late tip and AT2 cells). (E) Sections from control ( Nkx2.1-Cre; Stat3 +/fx ) and mutant ( Nkx2.1-Cre; Stat3 Δ/fx ) lungs with and without IL6. n =9 Nkx2.1-Cre; Stat3 Δ/fx lungs analysed in three independent experiments. Top panels: green, pro-SFTPC; red, LAMP3. Lower panels: red, pSTAT3; white, E-CAD (epithelium). Blue, DAPI. Dashed line, edge of lung. Scale bars: 50 μm in A,D; 100 μm in C,E.

Article Snippet: Primary antibodies: acetylated tubulin (mouse, 1:3000, Sigma, T7451), CEBPA (rabbit, 1:500, Santa Cruz, sc-61), cleaved caspase 3 (rabbit, 1:100, Abcam, ab2302), E-CAD (rat, 1:1000, Invitrogen, 13-1900; or mouse, 1:1000, BD Biosciences, 610182), GFP (chick, 1:1000, Abcam, AB13970), GR (rabbit, 1:100, Santa Cruz, sc-1004), HOPX (rabbit, 1:50, Santa Cruz, sc-30216, clone FL-73), KI67 (mouse, 1:200, BD, 550609), LAMP3 (rat, 1:100, Dendritics, DDX0192, clone 1006F7.05), LIF (goat, 1:100, R&D Systems, AB-449-NA), LPCAT1 (rabbit, 1:500, Proteintech), PDPN (hamster, 1:1000, DSHB, 8.1.1), RFP (rabbit, 1:250, Rockland, 600-401-379), pro-SFTPC (rabbit, 1:500, Millipore, AB3786), SOX2 (goat, 1:250, Santa Cruz, sc-17320, clone Y-17), SOX9 (goat, 1:200, R&D Systems, AF3075), pSTAT3-Tyr705 (rabbit, 1:200, Cell Signaling, 9145), STAT5a (rabbit, 1:20, Abcam, ab7968).

Techniques: Activation Assay, Control, Mutagenesis

Journal: Journal of neurochemistry

Article Title: Recapitulation of cell signaling events associated with astrogliosis using the brain slice preparation.

doi: 10.1111/j.1471-4159.2006.04321.x

Figure Lengend Snippet: Fig. 2 Relative mRNA expression levels for various cytokines and chemokines in the striatal slice preparation. Striatal mRNA expression of monocyte chemoattractive protein-1 (MCP-1), leukemia inhibitory factor (LIF), oncostatin M (OSM), interleukin-6 (IL-6), ciliary neuro- trophic factor (CNTF), and glial fibrillary acidic protein (GFAP) was examined by reverse transcriptase-polymerase chain reaction using template specific primers with GAPDH serving as an internal control. Agarose gel analysis shows a band of expected size in each case (LIF 388 bp, MCP-1 303 bp, OSM 266 bp, IL-6 154 bp, CNTF 243 bp, GFAP 462 bp, GAPDH 276 bp). Lanes were loaded with RNA pre- pared from slices as follows: 0 m (frozen immediately), 30, 45, 60, and 75 m post-preparation. Representative gels from at least three inde- pendent experiments are displayed. mRNA expression levels for MCP-1, OSM, and IL-6 at 60 min were significantly increased over 0 min control (p < 0.05–0.01, one-way ANOVA). mRNA expression for LIF at 75 min was significantly increased over 0 min control (p < 0.01; one-way ANOVA). CNTF and GFAP did not differ significantly from 0 min control at any time point. These images were prepared in accordance with the Provisional Guide for Digital images (http:// www.nature.com/nature/authors/submissions/images/index.html).

Article Snippet: Recombinant mouse oncostatin M (OSM), anti-mouse leukemia inhibitory factor (LIF), and antimouse OSM antibody were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Slice Preparation, Reverse Transcription, Polymerase Chain Reaction, Control, Agarose Gel Electrophoresis

Journal: Journal of neurochemistry

Article Title: Recapitulation of cell signaling events associated with astrogliosis using the brain slice preparation.

doi: 10.1111/j.1471-4159.2006.04321.x

Figure Lengend Snippet: Fig. 4 Phosphorylation of STAT3(tyr705) is increased by addition of the gp130 cytokines, leukemia inhibitory factor (LIF) and oncostatin M (OSM), to the striatal slice preparation. These effects are antagonized by addition of neutralizing antibodies directed against these cytokines. Immunoblot analysis of P-STAT3(tyr705) in striatal slices following incubation in buffer alone, 100 lmol/L leukemia inhibitory factor (LIF), 100 lmol/L OSM, or co-incubation with 50 lg a-LIF or 50 lg a-OSM for 45 min. These images were prepared in accordance with the Provisional Guide for Digital images (http://www.nature.com/nature/ authors/submissions/images/index.html).

Article Snippet: Recombinant mouse oncostatin M (OSM), anti-mouse leukemia inhibitory factor (LIF), and antimouse OSM antibody were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Slice Preparation, Western Blot, Incubation

Journal: Journal of neurochemistry

Article Title: Recapitulation of cell signaling events associated with astrogliosis using the brain slice preparation.

doi: 10.1111/j.1471-4159.2006.04321.x

Figure Lengend Snippet: Fig. 5 Mechanical damage associated with preparation of striatal brain slices results in recapitulation of cell signaling events associated with astrogliosis. For example, slice-induced or exogenously applied leukemia inhibitory factor (LIF) or oncostatin M (OSM) results in the enhanced phosphorylation of STAT3(tyr705), an event known to be associated with activation of astrocytes. LIF, OSM and other mediat- iors may emanate from the damaged cells, activated microglia or even the astrocyte itself. The effects of LIF and OSM on phosphorylation of STAT3(tyr705) can be blocked by addition of neutralizing antibodies (a- LIF or a-OSM) to the slice.

Article Snippet: Recombinant mouse oncostatin M (OSM), anti-mouse leukemia inhibitory factor (LIF), and antimouse OSM antibody were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Activation Assay