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: Cancer discovery

Article Title: IL-1-induced JAK/STAT signaling is antagonized by TGF-β to shape CAF heterogeneity in pancreatic ductal adenocarcinoma.

doi: 10.1158/2159-8290.CD-18-0710

Figure Lengend Snippet: A. qPCR analysis of iCAF (Il1a, Il6, Lif, Cxcl1 and Csf3) and myCAF (Acta2 and Ctgf) markers in PSCs cultured in Matrigel in control media in the presence or absence of 1 ng/mL mouse IL-1α for 4 days. Results show mean ± SEM of 2 biological replicates. *P<0.05, **P<0.01, ***P<0.001, paired Student’s t test. B. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence of a neutralizing antibody targeting IL-1α or an IgG control for 4 days. Results show mean ± SEM of 6 biological replicates. *P<0.05, **P<0.01, paired Student’s t test. C. Proliferation curves of PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence of a neutralizing antibody targeting IL-1α or an IgG control. Results show mean ± SEM of 3 biological replicates. **P<0.01, ***P<0.001, unpaired Student’s t test calculated for the last time point. D. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in PSCs cultured in Matrigel in transwell with Rosa26-targeted controls or IL-1α knockout (KO) tumor organoids for 4 days. Results show mean ± SEM of 9 and 11 biological replicates, respectively. ***P<0.001, paired Student’s t test. E. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in Rosa26-targeted controls and IL-1R1 knockout PSCs cultured in Matrigel in transwell with tumor organoids for 4 days. Results show mean ± SEM of 7 biological replicates. *P<0.05, ***P<0.001, paired Student’s t test. F. Proliferation curves of Rosa26-targeted controls and IL-1α knockout tumor organoids. Results show mean ± SD (standard deviation) of 5 technical replicates. *P<0.05, **P<0.01, unpaired Student’s t test calculated for the last time point. G. Tumor volume analysis based on ultrasound measurements of orthotopically grafted organoids (OGOs) following ~3 weeks from transplantation of Rosa26-targeted controls and IL-1α knockout tumor organoids in nu/nu mice. Results show mean ± SEM of 14 (control OGOs), 7 (1C or 1D OGOs) and 8 (1E OGOs) biological replicates. **P<0.01, ***P<0.001, unpaired Student’s t test. H. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in CAFs sorted from OGOs derived from transplantation of Rosa26-targeted controls and IL-1α knockout tumor organoids in nu/nu mice. Results show mean ± SEM of 4 (organoids), 12 (control OGOs) and 19 (IL-1α knockout OGOs) biological replicates. Different symbols identify the 3 knockout lines. *P<0.05, **P<0.01, ***P<0.001, paired Student’s t test. I. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2, Ctgf and Il1r1 in CAFs sorted from tumors derived by orthotopic transplantation of 3 tumor organoid lines in IL-1R1 knockout or C57BL/6J controls. Results show mean ± SEM of 9 biological replicates. *P<0.05, **P<0.01, ***P<0.001, paired Student’s t test. J. Quantification of Ly6C- myCAF/Ly6C+ iCAF ratio in tumors derived by orthotopic transplantation of 2 tumor organoid lines in B6J or IL-1R1 knockout hosts, as assessed by flow cytometry. Results show mean ± SEM of 5 biological replicates. **P<0.01, unpaired Student’s t test.

Article Snippet: Cells were treated with 0.1 or 1 ng/mL mouse (400-ML-005/CF, R&D Systems) or human (200-LA-002/CF, R&D Systems) IL-1α, 1 ng/mL mouse IL-1β (401-ML-005/CF, R&D Systems), 10 ng/mL mouse TNF-α (410-MT-010/CF, R&D Systems), 20 ng/mL mouse (7666-MB-005/CF, R&D systems) or human TGF-β1 (T7039-2UG, Sigma), 500 nM JAK inhibitor AZD1480 (S2162, Selleck Chem), 30 μM IKK-β inhibitor ML102B, 1 μM A83-01 (2939, Tocris Bioscience), 3 μg/mL IL-1α neutralizing antibody (MAB4001, R&D Systems) or an IgG control (400902, Biolegend), 5 μg/mL TNF-α neutralizing antibody (11969S, Cell signaling) or an IgG control (sc-2027, Santa Cruz), 3.4 μg/mL LIF neutralizing antibody (AF449, R&D Systems) or an IgG control (AB-108-C, R&D), 3.8 μg/mL G-CSF (MAB414, R&D Systems) or IL-6 (MAB406, R&D Systems) neutralizing antibodies or an IgG control (MAB005, R&D).

Techniques: Cell Culture, Control, Knock-Out, Standard Deviation, Transplantation Assay, Derivative Assay, Flow Cytometry

Journal: Cancer discovery

Article Title: IL-1-induced JAK/STAT signaling is antagonized by TGF-β to shape CAF heterogeneity in pancreatic ductal adenocarcinoma.

doi: 10.1158/2159-8290.CD-18-0710

Figure Lengend Snippet: A. qPCR analysis of iCAF (Il1a, Il6, Lif, Cxcl1 and Csf3) and myCAF (Acta2 and Ctgf) markers in PSCs cultured in Matrigel in control media in the presence or absence of 1 ng/mL mouse IL-1α for 1 h. Results show mean ± SEM of 2 biological replicates. *P<0.05, **P<0.01, paired Student’s t test. B. Western blot analysis of p-JAK1, JAK1, p-JAK2, JAK2, p-STAT1, STAT1, p-STAT3 and STAT3 in PSCs cultured in Matrigel with control media in the presence or absence of 1 ng/mL mouse IL-1α for 4 days. Loading control, ACTIN. C. Western blot analysis of p-JAK1, JAK1, p-JAK2, JAK2, p-STAT1, STAT1, p-STAT3 and STAT3 in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media (CM) in the presence of a neutralizing antibody targeting IL-1α or an IgG control for 4 days. Loading control, ACTIN. D. Western blot analysis of p-JAK1, JAK1, p-JAK2, JAK2, p-STAT1, STAT1, p-STAT3 and STAT3 in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media (CM) from Rosa26-targeted controls or IL-1α knockout tumor organoids for 4 days. Loading control, ACTIN. E. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media in the presence of a neutralizing antibody targeting LIF or an IgG control for 4 days. Results show mean ± SEM of 6 biological replicates. *P<0.05, **P<0.01, paired Student’s t test. F. Western blot analysis of p-JAK1, JAK1, p-JAK2, JAK2, p-STAT1, STAT1, p-STAT3 and STAT3 in PSCs cultured in Matrigel with control media or tumor organoid-conditioned media (CM) in the presence of a neutralizing antibody targeting LIF or an IgG control for 4 days. Loading control, ACTIN. G. qPCR analysis of Il1a, Il6, Lif, Cxcl1, Csf3, Acta2 and Ctgf in LIF knockout PSCs compared to Rosa26-targeted controls cultured in Matrigel in transwell with tumor organoids for 4 days. Results show mean ± SEM of 4 biological replicates. *P<0.05, **P<0.01, ***P<0.001, paired Student’s t test.

Article Snippet: Cells were treated with 0.1 or 1 ng/mL mouse (400-ML-005/CF, R&D Systems) or human (200-LA-002/CF, R&D Systems) IL-1α, 1 ng/mL mouse IL-1β (401-ML-005/CF, R&D Systems), 10 ng/mL mouse TNF-α (410-MT-010/CF, R&D Systems), 20 ng/mL mouse (7666-MB-005/CF, R&D systems) or human TGF-β1 (T7039-2UG, Sigma), 500 nM JAK inhibitor AZD1480 (S2162, Selleck Chem), 30 μM IKK-β inhibitor ML102B, 1 μM A83-01 (2939, Tocris Bioscience), 3 μg/mL IL-1α neutralizing antibody (MAB4001, R&D Systems) or an IgG control (400902, Biolegend), 5 μg/mL TNF-α neutralizing antibody (11969S, Cell signaling) or an IgG control (sc-2027, Santa Cruz), 3.4 μg/mL LIF neutralizing antibody (AF449, R&D Systems) or an IgG control (AB-108-C, R&D), 3.8 μg/mL G-CSF (MAB414, R&D Systems) or IL-6 (MAB406, R&D Systems) neutralizing antibodies or an IgG control (MAB005, R&D).

Techniques: Cell Culture, Control, Western Blot, Knock-Out