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: Nature Communications

Article Title: Neonatal Wnt-dependent Lgr5 positive stem cells are essential for uterine gland development

doi: 10.1038/s41467-019-13363-3

Figure Lengend Snippet: Pre-pubertal Lgr5 high uterus cells behave as Wnt-regulated stem/progenitor cells in ex vivo organoid culture assays. a Representative image of a developing uterine organoid generated from a single EPCAM + GFP high cell isolated from a P14 Lgr5-2A-EGFP uterus. b H&E and IHC for K8 on a Lgr5 high cell-derived organoid. c Endogenous EGFP fluorescence on a Lgr5 high cell-derived organoid. Yellow arrowheads highlight the heterogenous expression of Lgr5-EGFP within individual organoids. Scale bars, 50 μm. d Organoids generated from single EPCAM + GFP high cells and EPCAM + GFP neg cells (image representative of five replicates). Scale bars, 1000 μm. e Quantification of organoid formation efficacy from single EPCAM + GFP high and EPCAM + GFP neg cells. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t -test. *** P < 0.001. f Passage frequency of organoids derived from single EPCAM + GFP high and EPCAM + GFP neg cells. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t -test ( P = 7E−07). g Experimental strategy to ablate Lgr5 + cells in vitro. h Organoid cultures derived from Lgr5-DTR-EGFP and wild-type (control) EPCAM + uterus cells following Lgr5 + cell ablation by DT treatment in vitro (image representative of five replicates). Scale bars, 200 μm. i Outgrowth efficiency of organoids derived from Lgr5-DTR-EGFP and wild-type mouse EPCAM + uterus cells following DT treatment in vitro. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t -test ( P = 4E−06). j Organoids derived from wild-type EPCAM + uterus cells cultured under low Wnt3a and low Wnt3a + CHIR conditions (image representative of five replicates). Scale bars, 200 μm. k The average proportions of organoid types under low Wnt3a, low Wnt3a + CHIR and high Wnt3a conditions. Data from five independent experiments are presented. Data were tested for significance by a chi-square test (Low Wnt3a vs. Low Wnt3a + CHIR: P = 2E−17, Low Wnt3a vs. High Wnt3a: P = 2E−08). l QPCR analysis of Lgr5 and Fzd10 expression on organoids generated under each culture condition. Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t -test ( Lgr5 : Low Wnt3a vs. Low Wnt3a + CHIR: P = 0.002, Low Wnt3a vs. High Wnt3a: P = 0.012, Fzd10 : Low Wnt3a vs. Low Wnt3a + CHIR: P = 0.018, Low Wnt3a vs. High Wnt3a: P = 0.027). m QPCR analysis of GE markers ( Foxa2, Lif, Prss28 ) and LE markers ( Wnt7a, Scnn1a, Irg1 ) expression on organoids generated under each culture condition Data from five independent experiments are presented as mean ± s.e.m. Data were tested for significance using unpaired two-tailed t -test ( Foxa2 : P = 9E−04, Lif : P = 0.006, Prss28 : P = 0.54, Wnt7a : P = 0.46, Scnn1a : P = 0.49, Irg1 : P = 5E−06). n , o Immunostaining for Foxa2, Lif, and Ki67 on round and vacuolated-type organoids. Scale bars, 50 μm. Red arrows indicate Foxa2-positive cells (image representative of three independent mice). *** P < 0.001, ** P < 0.01, * P < 0.05, N.S., not significant.

Article Snippet: The following primary antibodies were employed: mouse anti-Lim1 (1:100; DSHB, 4F2), rabbit anti-Foxa2 (1:400; Cell Signaling, 8186), rabbit anti-K8 (1:400; Abcam, ab53280), rabbit anti-vimentin (1:1000; Abcam, ab92547), mouse anti-E-cadherin (1:200; BD Transduction Laboratories, 610181), rabbit anti-cleaved Caspase3 (1:200; Cell Signaling, 9661), rabbit anti-Ki67 (1:200; ThermoFisher, MA5-14520), rabbit anti-LIF (1:200; Origene, TA321468), mouse anti-Ki67 (1:200; BD Transduction Laboratories, 550609), chicken anti-GFP (1:100; Abcam, ab13970), rabbit anti-GFP (1:200; Cell Signaling, 2956S), rabbit anti-RFP (1:200; Rockland, 600-401-379), mouse anti-RFP (1:100; Abcam, ab125244).

Techniques: Ex Vivo, Generated, Isolation, Derivative Assay, Fluorescence, Expressing, Two Tailed Test, In Vitro, Cell Culture, Immunostaining