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: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of genes regulated by leukemia-inhibitory factor in the mouse uterus at the time of implantation.

doi: 10.1210/me.2004-0110

Figure Lengend Snippet: Fig. 1. Regulation of Uterine IGFBP3 mRNA Expression by LIF Recombinant LIF was injected into one horn and PBS into the other horn of LIF/ females at 84 h of pseudopregnancy (n 10). IGFBP3 mRNA expression levels were determined in total uterine RNA isolated 12 h later by real-time RT-PCR. Values for IGFBP3 are normalized to 18S rRNA for each horn and expressed in arbitrary units relative to the level of the same gene in a standard RNA produced from mouse uterus. LIF treatment (solid bars) produced increased IGFBP3 expression in nine of 10 animals compared with the paired PBS-treated uterine horn (open bars). The mean increase was 2.3-fold and was statistically significant (P 0.02, paired Mann-Whitney).

Article Snippet: Two ip injections each of 5 g recombinant human LIF (R&D systems 250-LF) in PBS were given at 0900 h and 1600 h on d 4 of pregnancy, and the animals were allowed to proceed to term.

Techniques: Expressing, Recombinant, Injection, Isolation, Quantitative RT-PCR, Produced, MANN-WHITNEY

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of genes regulated by leukemia-inhibitory factor in the mouse uterus at the time of implantation.

doi: 10.1210/me.2004-0110

Figure Lengend Snippet: Fig. 6. LIF Regulation of Amphiregulin and IRG1 in the Uterus Recombinant LIF was injected into one horn and PBS into the other horn of LIF/ females at 84 h of pseudopreg- nancy (n 10, same animals as shown in Fig. 1). Amphiregu- lin and IRG1 mRNA expression levels were determined in total uterine RNA isolated 12 h later by real-time RT-PCR. Values for each mRNA species are normalized to 18S rRNA for each horn and expressed in arbitrary units relative to the level of the same gene in a standard RNA produced from mouse uterus. LIF treatment (solid bars) increased both am- phiregulin and IRG1 expression in 10 of 10 animals compared with the corresponding PBS-treated horn (open bars). The mean increase for amphiregulin was 7.3-fold and for IRG1 was 3.7-fold and was statistically significant for either gene (P 0.002, paired Mann-Whitney test).

Article Snippet: Two ip injections each of 5 g recombinant human LIF (R&D systems 250-LF) in PBS were given at 0900 h and 1600 h on d 4 of pregnancy, and the animals were allowed to proceed to term.

Techniques: Recombinant, Injection, Expressing, Isolation, Quantitative RT-PCR, Produced, MANN-WHITNEY

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

Journal: Nature Communications

Article Title: The human nucleoporin Tpr protects cells from RNA-mediated replication stress

doi: 10.1038/s41467-021-24224-3

Figure Lengend Snippet: a U-2 OS cells were transfected using siRNAs and either nontreated (Mock) or treated with 2 mM hydroxyurea (HU) for 4 h. RPA, loading control. Data are representative of three independent experiments. b U-2 OS cells were transfected using siRNAs and treated as in a . RPA, loading control. The experiment was performed twice yielding similar results. c U-2 OS cells were transfected using siRNAs. Tubulin, loading control. Data are representative of three independent experiments. d U-2 OS cells were transfected using siRNA targeting endogenous Tpr (siTPR #54), GANP (siGANP #87), or control siRNA (siCTRL). In parallel, cells were cotransfected with siRNA-resistant Tpr-GFP insensitive to siTPR #54. Tpr-GFP and GANP protein levels were analyzed by immunoblotting. Tubulin, loading control. The experiment was performed twice yielding similar results. e U-2 OS cells were transfected with siRNAs and levels of Tpr and GANP were assessed using immunofluorescence. Nuclei were stained with DAPI. The scale bar represents 7.5 µm. Data are representative of three independent experiments. f Expression of siRNA-resistant full-length Tpr-wt can rescue GANP protein level. U-2 OS cells were transfected with siRNA targeting endogenous Tpr (siTPR), and with siRNA-resistant Tpr-wt-GFP (TPR-wt). Levels of Tpr-wt-GFP and GANP were assessed using immunofluorescence. Nuclei, DAPI. The scale bar, 7.5 µm. Data are representative of three independent experiments. g Expressions of Tpr-N-terminal domain or Tpr-C-terminal domain are not able to rescue GANP protein level. U-2 OS cells were transfected with siRNA targeting endogenous Tpr (siTPR), and with Tpr-N-terminal-FLAG (TPR-N) or Tpr-C-terminal-FLAG (TPR-C) constructs. Levels of Tpr-FLAG and GANP were assessed using immunofluorescence. Nuclei, DAPI. The scale bar, 7.5 µm. Data are representative of three independent experiments. h Expression of siRNA-resistant full-length Tpr-NEBM (Nuclear Envelope Binding Mutant) is not able to rescue GANP protein level. U-2 OS cells were transfected with siRNA targeting endogenous Tpr (siTPR), and with siRNA-resistant Tpr-NEBM1-GFP (NEBM1) construct carrying L458P/M489P double amino acid substitution in Tpr nuclear envelope binding domain. Levels of Tpr-NEBM1-GFP and GANP were assessed using immunofluorescence. Nuclei, DAPI. The scale bar, 7.5 µm. Data are representative of three independent experiments. Source data for a – d are provided in the file.

Article Snippet: The following primary antibodies were used: ATR (Cell Signaling, #2790, 1:1000), phospho-ATR (Thr1989) (Cell Signaling, #58014, 1:1000), Chk1 (Santa Cruz Biotechnology, #sc-8408, 1:1000), phospho-Chk1 (Ser345) (Cell Signaling, #2348, 1:1000), phospho-Chk1 (Ser317) (Cell Signaling, #12302, 1:1000), cyclin A2 (Abcam, #ab16726, 1:000), GANP (Abcam, #ab113295, 1:1000), GANP (Sigma-Aldrich, #HPA021527, 1:1000), GFP (Amsbio, TP401, 1:2500), MATR3 (Bethyl, #A300-591A, 1:1000), PTBP1 (Thermo Fisher Scientific, #32-4800, 1:000), RPA32 (Abcam, #ab16855, 1:2000), phospho-RPA32 (S4/S8) (Bethyl, #A300-245A, 1:1000), SUGP2 (Sigma-Aldrich, #HPA061111, 1:1000), Tpr (Sigma-Aldrich, #HPA019661, 1:1000), and tubulin (Sigma-Aldrich, #T5168, 1:5000).

Techniques: Transfection, Control, Western Blot, Immunofluorescence, Staining, Expressing, Construct, Binding Assay, Mutagenesis

Journal: BMC Genomics

Article Title: JAK / STAT3 regulated global gene expression dynamics during late-stage reprogramming process

doi: 10.1186/s12864-018-4507-2

Figure Lengend Snippet: Restoring JAK / STAT3 Activity Promotes DNA Demethylation of Multi-Pluripotency Loci in pre-iPSCs. a Jaki-S2 pre-iPSC colonies cultured and expanded in the presence of LIF plus Jaki (LIF+/Jaki+), LIFAb (LIF+/LIFAb+), or LIF-only (LIF+) for two weeks. Bar = 625 um. b FACS analysis for GFP+ and GFP- cells in expanded Jaki-S2 pre-iPSCs cultured in LIF containing medium supplemented with Jaki, LIFAb, or LIF-only condition for 3 weeks. Numbers represent mean ± SD from three independent experiments. c DNA methylation of Oct4 promoter region measured by bisulfite sequencing for samples described in b . Filled and open circles represent methylated and unmethylated CpGs, respectively. The percentage of total methylated CpGs for the analyzed region was given on top of each dataset. d DNA methylation of IG-DMR region measured by bisulfite sequencing for samples described in b . Filled and open circles represent methylated and unmethylated CpGs, respectively. The percentage of total methylated CpGs for the analyzed region was given on top of each dataset. e The proposed model of JAK/STAT3 in promoting naïve-state pluripotency in reprogramming. LIF activated JAK/STAT3 suppresses Hdacs and stimulates Hats / Kats , Tet1 , Dppa3 , and PRC2 genes for open chromatin formation in pluripotent and gametogenic loci, promotes their full activation. Dppa3 and PRC2 inhibit de novo methylation of pluripotent loci by Dnmt3s . On the other hand, STAT3 activated Dnmt3s inhibit commitment genes and the OKSM transgenes, the silencing of which are needed for complete reprogramming. This leads to the complete activation of pluripotent circuitry and establishment of naïve-state iPSCs

Article Snippet: LIF antibody (LIFAb) was from Santa Cruz (Santa Crutz, CA, USA).

Techniques: Activity Assay, Cell Culture, DNA Methylation Assay, Methylation Sequencing, Methylation, Activation Assay