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) as well as treatment with recombinant human LIF protein (rhLIF, 100 ng/ml for 6 h; right panel enhanced glucose uptake in breast cancer cell lines, including MCF7, MDA-MB 231, T47D, MDA-MB 468, ZR-75-1 and SKBR3 cells as determined by measuring the uptake of 3 H-2-DG in cells. The overexpression of LIF in these cells was shown in Supplementary Fig . B Knockdown of endogenous LIF by two different shRNAs reduced glucose uptake in MCF7 and MDA-MB 231 cells. The knockdown of LIF expression in these cells was shown in Supplementary Fig . C Ectopic LIF expression increased glucose uptake in the xenograft tumors formed by MDF7 and MDA-MB 231 cells as determined by measuring the uptake of 3 H-2-DG in tumor tissues. D . High LIF expression was associated with the increased 18 F-FDG uptake in human breast tumors. Left panels: representative PET scan data and images of LIF IHC staining. SUV: standardized uptake value. In A – C , n = 3/group; in D , n = 18 for total patient samples. *: p < 0.05; **: p < 0.01; ***: p < 0.001; unpaired Student’s t -test for A – C ; correlation in D was calculated by Spearman’s Rho correlation analysis.

Article Snippet: The Taqman primers for human LIF (Hs00171455_m1) and human actin (Hs99999903_m1) were purchased from Applied Biosystems.

Techniques: Expressing, Recombinant, Over Expression, Knockdown, Immunohistochemistry

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: The Taqman primers for human LIF (Hs00171455_m1) and human actin (Hs99999903_m1) were purchased from Applied Biosystems.

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 2-DG preferentially inhibited the proliferation of breast cancer cells with ectopic LIF expression compared with control cells transduced with control vectors. MCF7, MDA-MB 231, and T47D cells with or without ectopic LIF expression were treated with 2-DG at different concentrations. B 2-DG treatment exhibited a more pronounced inhibitory effect on growth of xenograft tumors formed by MCF7 and MDA-MB 231 cells with ectopic LIF expression than those formed by control cells. Mice with xenograft tumors (~50 mm 3 ) were administered with 2-DG (i.p., 1 g/kg mice, every two days for two weeks). Arrows represent the start of 2-DG treatment. Data are presented as mean ± SD. In A , n = 3/group; in B , n ≥ 5/group. * p < 0.05; ** p < 0.01; *** p < 0.001; 2-way ANOVA followed by Student’s t -test.

Article Snippet: The Taqman primers for human LIF (Hs00171455_m1) and human actin (Hs99999903_m1) were purchased from Applied Biosystems.

Techniques: Expressing, Control, Transduction

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: The Taqman primers for human LIF (Hs00171455_m1) and human actin (Hs99999903_m1) were purchased from Applied Biosystems.

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

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: Endogenous Glut1 was knocked down by shRNA in MCF7, MDA-MB 231, and T47D cells with or without ectopic LIF expression. A , B Glucose uptake ( A ) and lactate production ( B ) were measured in cells. C The efficiency of Glut1 knockdown was determined at mRNA levels by qPCR. D Knockdown of Glut1 greatly inhibited the promoting effect of LIF on xenograft tumor growth. MCF7 and MDA-MB 231 cells with or without ectopic LIF expression along with or without Glut1 knockdown were employed for xenograft tumorigenesis assays. E Knockdown of Glut1 greatly blocked the promoting effect of LIF on glucose uptake in xenograft tumors formed by MCF7 and MDA-MB 231 cells. Glucose uptake was measured in xenograft tumors described in D . Data are presented as mean ± SD. In A , B , C & E : n = 3/group; in D : n = 6/group. * p < 0.05; ** p < 0.01; *** p < 0.001; unpaired Student’s t -test for A , B , C , E , and 2-way ANOVA followed by Student’s t -test for D.

Article Snippet: The Taqman primers for human LIF (Hs00171455_m1) and human actin (Hs99999903_m1) were purchased from Applied Biosystems.

Techniques: shRNA, Expressing, Knockdown

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 LIF activated the AKT signaling, which can be blocked by MK2206, an AKT inhibitor (upper panel), or Wortmannin (Wort), a PI3K inhibitor (lower panel), in MCF7, MDA-MB 231 and T47D cells. The levels of p-AKT Ser473 (p-AKT), which reflect the activity of AKT were measured by Western-blot assays. B Blocking AKT signaling by MK2206 (left) or Wortmannin (right) largely abolished the promoting effect of LIF on Glut1 PM translocation in breast cancer cells. C , D Blocking AKT signaling by expression of dominant-negative AKT (AKT-DN) largely abolished the promoting effect of LIF on Glut1 PM translocation in breast cancer cells. Cells were transfected with vectors expressing AKT-DN. The levels of p-AKT and total AKT ( C ), as well as Glut1 PM translocation ( D ), were determined by Western-blot assays. Uncropped Western-blot images are shown in Supplementary Fig .

Article Snippet: The Taqman primers for human LIF (Hs00171455_m1) and human actin (Hs99999903_m1) were purchased from Applied Biosystems.

Techniques: Activity Assay, Western Blot, Blocking Assay, Translocation Assay, Expressing, Dominant Negative Mutation, Transfection

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 , B Blocking AKT activation by MK2206 (1 µM for 12 hours) ( A , left panel), Wortmannin (2 µM for 6 h) ( A , right panel) or AKT-DN expression ( B ) largely abolished the promoting effect of LIF on glucose uptake in breast cancer cell lines. C , D . Treatment with MK2206 ( C , left panel), Wortmannin ( C , right panel) or AKT-DN expression ( D ) largely abolished the promoting effect of LIF on lactate production in breast cancer cell lines. E . Blocking AKT signaling by Wortmannin greatly inhibited the promoting effect of LIF on xenograft tumor growth. Mice with xenograft tumors (~50 mm 3 ) were administered with Wortmannin ( i.p ., 1.5 mg/kg mice, daily for two weeks). F Wortmannin treatment largely abolished the promoting effect of LIF on glucose uptake in xenograft tumors. Glucose uptake was measured in xenograft tumors described in E . G Schematic illustration of the role of LIF in increasing glycolysis, which in turn promotes breast tumorigenesis. Data are present as mean ± SD. For A – D , F : n = 3/group; For E : n ≥ 6/group. * p < 0.05; ** p < 0.01; *** p < 0.001; unpaired Student’s t -test for A – D , F , and 2-way ANOVA followed by Student’s t -test for E .

Article Snippet: The Taqman primers for human LIF (Hs00171455_m1) and human actin (Hs99999903_m1) were purchased from Applied Biosystems.

Techniques: Blocking Assay, Activation Assay, Expressing

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: 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: 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