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: Frontiers in Immunology

Article Title: The dysregulation of leukemia inhibitory factor and its implications for endometriosis pathophysiology

doi: 10.3389/fimmu.2023.1089098

Figure Lengend Snippet: LIF treatment in a mouse model of endometriosis alters the local peritoneal immune response. (A) Gating strategy for flow cytometric analysis of myeloid (B–F) and lymphoid (G–J) markers on immune cells from the PF of mice injected i.p with PBS (white bars) or rmLIF (grey bars; 300ng, 1 µ g) for 14 days. LPM gated as: single cells, live, SSC low , CD11b + , F4/80 hi , MHCII low . SPM gated as: single cells, live, SSC low , CD11b + , F4/80 mid , MHCII hi . Tregs gated as: single cells, live, SSC low , CD11b - , F4/80 - , MHCII - , CD3 + , CD4 + , CD25 + , FOXP3 + . Results reflect duplicate experiments- one per rmLIF dosage. Analysis performed as unpaired Student’s T-test, *P<0.05, **P<0.01. LPM, large peritoneal macrophages, SPM, small peritoneal macrophages.

Article Snippet: To understand the influence of LIF on endometrial lesion growth and immune cell populations, mice received daily intraperitoneal (i.p) injections of either PBS (control; n=6) or recombinant mouse LIF (rmLIF; n=6; 8878-LF-100/CF, R&D Systems) for 14 days.

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: The dysregulation of leukemia inhibitory factor and its implications for endometriosis pathophysiology

doi: 10.3389/fimmu.2023.1089098

Figure Lengend Snippet: LIF treatment in a mouse model of endometriosis alters the peripheral immune response. (A) Gating strategy for flow cytometric analysis of myeloid (B) and lymphoid markers (C–F) on immune cells from the spleen of mice injected i.p with PBS (white bars) or rmLIF (grey bars; 300ng, 1µg) for 14 days. Tregs gated as: single cells, live, SSC low , CD11b - , F4/80 - , MHCII - , CD3 + , CD4 + , CD25 + , FOXP3 + . Results reflect duplicate experiments- one per rmLIF dosage. Analysis performed as unpaired Student’s T-test, *P<0.05, **P<0.01.

Article Snippet: To understand the influence of LIF on endometrial lesion growth and immune cell populations, mice received daily intraperitoneal (i.p) injections of either PBS (control; n=6) or recombinant mouse LIF (rmLIF; n=6; 8878-LF-100/CF, R&D Systems) for 14 days.

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: The dysregulation of leukemia inhibitory factor and its implications for endometriosis pathophysiology

doi: 10.3389/fimmu.2023.1089098

Figure Lengend Snippet: LIF treatment did not alter lesion growth or proliferation in a mouse model of endometriosis. I.p injections of PBS (white bars) or rmLIF (grey bars; 300ng, 1µg) were administered to C57BL/6 mice (n=6 for all groups) for 14 days, one week after endometriosis inducing surgery. Endometriosis-like lesions were collected upon sacrifice and subjected to IHC for markers of proliferation-Ki67 (A, B) and angiogenesis-CD31 (E, F) , as well as LIF (I, J) and LIFR (M, N) . Representative stain analysis provided for each marker from both the PBS and LIF treatment groups. Ki67 was analyzed as percent of cells expressing Ki67 over the total cell number as detected by a cytonuclear algorithm (C, D) , while all other stains (CD31, LIF, LIFR) were analyzed by percent area quantification of stain (G, H, K, L, O, P) . No statistical differences were detected across the four stains. Analysis performed as unpaired Student’s T-test. Scale bar 100µm.

Article Snippet: To understand the influence of LIF on endometrial lesion growth and immune cell populations, mice received daily intraperitoneal (i.p) injections of either PBS (control; n=6) or recombinant mouse LIF (rmLIF; n=6; 8878-LF-100/CF, R&D Systems) for 14 days.

Techniques: Staining, Marker, Expressing

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: VEGF-induced vascular permeability is reduced upon CRISPR/Cas9-mediated knockout of Stat3 in zebrafish. (A) VEGF-inducible zebrafish were crossed to Stat3 +/− (heterozygous) zebrafish to generate VEGF-inducible; Stat3 +/− double transgenic fish, which were intercrossed to generate VEGF-inducible; Stat3 −/− (KO) zebrafish. (B) CRISPR/Cas9-generated Stat3 KO zebrafish (bottom) display no overt vascular defects relative to wild-type (WT) zebrafish (top). The vascular system of 3 days post-fertilization (dpf) zebrafish was visualized by microangiography with 2000 kDa FITC-dextran. Representative images of at least three zebrafish per group are shown. Scale bars: 100 μm. (C) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf Stat3 +/+ (negative controls without VEGF induction; left) , VEGF-induced, Stat3 +/+ (middle) and VEGF-induced, Stat3 −/− (right) zebrafish. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at room temperature (RT). Scale bars: 50 μm. (D) Quantitative analysis of vascular permeability upon VEGF stimulation in WT Stat3 +/+ ( n =30) and KO Stat3 −/− ( n =9) zebrafish. Mean±s.e.m., unpaired, two-tailed Student's t -test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, CRISPR, Knock-Out, Transgenic Assay, Generated, Marker, Microscopy, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Endothelial cell-specific STAT3 knockout mice exhibit decreased VEGF-induced permeability. (A) Images of footpads from WT and endothelial cell-specific STAT3 knockout (STAT3 ECKO ) mice following tail vein injection with 1% Evans Blue dye and human recombinant VEGF-165 protein (2.5 µg/ml; left footpads) or PBS vehicle (right footpads) being injected into the root of the footpad. (B,C) Quantitation of Evans Blue leakage in Tie2-Cre negative; STAT3 flox/flox (WT) and Tie2-Cre positive; STAT3 flox/flox (STAT3 ECKO ) mice. n =7 mice in WT group and n =6 mice in STAT3 ECKO group. Each mouse was injected with PBS on the right anterior and posterior footpads and VEGF on the left anterior and posterior footpads. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. A.U., arbitrary units.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Knock-Out, Permeability, Injection, Recombinant, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Pharmacological inhibition of STAT3 stabilizes endothelial barrier integrity following VEGF stimulation in human endothelial cells. (A) Serum-starved human umbilical vein endothelial cells (HUVECs) were pretreated with DMSO (vehicle control) for 1 h, 30 µM AQ for 4 h, or 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 2 or 5 min. Lysates were immunoblotted. Densitometry was performed, and the values below the rows of bands represent the ratio of phosphorylated protein to respective total protein. (B) Human VEGF-165 recombinant protein (VEGF; 25 ng/ml) stimulation of HUVECs promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows; left column; DMSO vehicle control pretreatment for 1 h prior to VEGF stimulation). ZO-1 organization is maintained upon pretreatment with 30 μM AQ for 4 h (magenta arrows; middle column) or 10 μM PYR for 1 h (magenta arrows; right column) prior to VEGF stimulation. Nuclei were stained with DAPI (blue). (C) Serum-starved human pulmonary artery endothelial cells (HPAECs) were pretreated with 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 5 or 30 min. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained when HPAECs were pretreated with PYR (magenta arrows). Nuclei were stained with DAPI (blue). (D) VEGF (25 ng/ml) stimulation of human lung microvascular endothelial cells (HMVEC-Ls) promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained upon pretreatment with 20 μM PYR for 6 h prior to VEGF stimulation (magenta arrows). Nuclei were stained with DAPI (blue). At least two biological replicates were performed for each experiment depicted in A-D. Scale bars: 20 µm.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Inhibition, Recombinant, Staining

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Suppression of STAT3 activity by pyrimethamine (PYR) inhibits VEGF-induced vascular permeability in zebrafish and mice. (A) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf zebrafish without induced VEGF pretreated with DMSO ( n =6) or 25 μM PYR ( n =5) or 3 dpf zebrafish with induced VEGF pretreated with DMSO ( n =4) or 25 μM PYR ( n =9) for 3 days. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at RT. Scale bars: 50 μm. (B) The quantitative analysis of vascular permeability without VEGF stimulation or upon VEGF stimulation in zebrafish pretreated with DMSO or PYR. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. (C) Representative images of footpads from mice treated with vehicle or PYR following tail vein injection with 1% Evans Blue and footpad injection of VEGF (2.5 μg/ml) or PBS vehicle. (D) Quantitation of Evans Blue dye leakage in C57BL/6 WT mice treated with vehicle or PYR. n =9 mice in the vehicle group and n =7 mice in the PYR group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Activity Assay, Permeability, Marker, Microscopy, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: JAK2 phosphorylates STAT3 to transduce VEGF/VEGFR-2 signaling and promote vascular permeability. (A) To perform a STAT3 GST pull-down of VEGFR-2 and JAK2, lysates of HUVECs stimulated with serum for 30 min were used as prey. GST fusion protein STAT3 expressed in 293F cells was used as bait. GST alone served as a negative control. Binding experiments were analyzed by SDS-PAGE and visualized by immunoblotting. GST-STAT3 and GST were each detected using an anti-GST antibody. Three biological replicates were performed and depicted findings are representative. (B) JAK2 phosphorylates STAT3 in vitro . In vitro kinase assays were performed using purified human STAT3 protein and kinase active JAK2 protein. The results shown here are representative of two independent experiments. (C) Representative images of footpads from C57BL/6 WT mice treated with vehicle or JAK2 inhibitor AG490. Following tail vein injection with 1% Evans Blue dye, human VEGF-165 protein (2.5 μg/ml) or PBS vehicle was injected into the root of the footpad. After 30 min, the mice were euthanized and the footpads were excised. (D) Quantitation of Evans Blue dye leakage in C57BL/6 mice treated with vehicle or AG490. n =4 mice per group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Two biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Transduction, Permeability, Negative Control, Binding Assay, SDS Page, Western Blot, In Vitro, Purification, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: STAT3 transcriptionally activates ICAM-1, a cell adhesion molecule that promotes vascular permeability. (A) Top: the pGL3-ICAM1-WT plasmid containing the human ICAM-1 promoter with a STAT3 binding site located at −115 to −107 bp. Bottom: the pGL3-ICAM1-SDM plasmid with a site-directed mutation (SDM) in the STAT3 binding site as indicated. (B) Dual luciferase assays were performed in HUVECs that were transfected with pGL3-ICAM1-WT or pGL3-ICAM1-SDM and empty vector or constitutively active STAT3. Firefly and Renilla luminescence was measured and plotted as a ratio. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. n =9 technical replicates. Depicted findings are representative of three independent experiments. (C) HUVECs that had been stably transduced with lentivirus encoding STAT3-specific shRNA or control shRNA were stimulated with human VEGF-165 protein (25 ng/ml) and the lysates were immunoblotted for ICAM1, p-STAT3 (Y705) and total STAT3. Depicted data are representative of three biological replicates. (D) RNA was harvested from VEGF; Stat3 +/+ or VEGF; Stat3 −/− 3 dpf embryos for quantitative PCR. stat3 transcripts are reduced in VEGF; Stat3 −/− ( n =5) compared to VEGF; Stat3 +/+ zebrafish ( n =7). Mean±s.e.m., unpaired, two-tailed Student's t -test. (E) The expression of icam-1 was assessed by real-time quantitative PCR using RNA derived from each zebrafish embryo in the absence of VEGF induction (Stat3 +/+ , n =3; Stat3 −/− , n =2) or 8 h following VEGF induction (Stat3 +/+ , n =4; Stat3 −/− , n =3) in the heat-inducible VEGF; Stat3 mutant zebrafish. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, Plasmid Preparation, Binding Assay, Mutagenesis, Luciferase, Transfection, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, Two Tailed Test, Expressing, Derivative Assay

Journal: Cell Transplantation

Article Title: Transplantation of Human Adipose Stem Cells Using Acellular Human Amniotic Membrane Improves Angiogenesis in Injured Endometrial Tissue in a Rat Intrauterine Adhesion Model

doi: 10.1177/0963689720952055

Figure Lengend Snippet: Histological analyses using H&E staining and LIF immunostaining. (A) The control, IUA, IUA-AHAM, and IUA-hASC-AHAM groups were analyzed by H&E staining. (B) The mean endometrial thickness and endometrial gland numbers in the four groups were quantitative analyzed. (C) Protein levels of LIF in the four groups were examined by immunohistochemical staining. (D) The LIF expression in the four groups was quantitative analyzed. NS: not significant, * P < 0.05, ** P < 0.01 by unpaired two-tailed Student’s t -test. The data are presented as the means ± SD. Black arrows indicate the glands. AHAM: acellular human amniotic membrane; hASC: human adipose stem cell; H&E: hematoxylin and eosin; IUA: intrauterine adhesion; IUA-AHAM: IUA treated with AHAM; IUA-hASC-AHAM: IUA treated with hASC-AHAM; LIF: leukemia inhibitory factor.

Article Snippet: Subsequently, the samples were incubated with the primary antibodies, including leukemia inhibitory factor (LIF) (1:175; Biorbyt, Cambridge, UK) and CD34 (1:7,000; Abcam, Cambridge, UK) at 4°C overnight.

Techniques: Staining, Immunostaining, Control, Immunohistochemical staining, Expressing, Two Tailed Test, Membrane

Journal: Neurobiology of disease

Article Title: Glycogen synthase kinase-3β inhibition enhances myelination in preterm newborns with intraventricular hemorrhage, but not recombinant Wnt3A

doi: 10.1016/j.nbd.2018.06.015

Figure Lengend Snippet: A) Representative immunofluorescence form corona radiata of cryosections from D3 pups stained with Iba-1 specific antibody. Insets (on right side of image) show high magnification views of the boxed area in the image. Iba1+ microglia (arrow) are more abundant in pups with IVH relative to controls without IVH and AR-A014418 treatment reduces their density. Scale bar, 50 μm. Bar charts are mean ± s.e.m. (n=5 each group). IVH elevates the density of Iba1+ microglia in both ganglionic eminences (lateral ventricular wall) and corona radiata; AR-A014418 treatment reduces their densities in both regions. B) mRNA expressions of TNFα, IL1β, IL 6, and LIF were elevated in IVH compared to controls with IVH at D3, and AR-A014418 treatment significantly reduced IL1β levels at D3. Data are mean ± s.e.m. (n=5 each group). *P<0.05, **P<0.01, ***P<0.001 pups with vs. without IVH. #P<0.05, ##P<0.01, ###P<0.001 vehicle vs. AR-A014418 treated pups with IVH.

Article Snippet: Their assay IDs were as follows: GAPDH (Oc03823402_g1), TNFα (Oc03397716_g1), IL1β (Oc03823250_s1), CNTF (Oc03397817_m1), LIF (Hs01055668_m1), IL-6 (Oc04097053_m1), Hes 5 (Mm00439311_g1), Hes 1 (APH49WG), GSk3b(Hs01047719_m1).

Techniques: Immunofluorescence, Staining

Journal: Neurobiology of disease

Article Title: Glycogen synthase kinase-3β inhibition enhances myelination in preterm newborns with intraventricular hemorrhage, but not recombinant Wnt3A

doi: 10.1016/j.nbd.2018.06.015

Figure Lengend Snippet: A) Representative immunofluorescence form corona radiata of cryosections from D3 pups stained with Iba-1 specific antibody. Insets (on right side of image) show high magnification views of the boxed area in the image. Scale bar, 50 μm. Bar charts are mean ± s.e.m. (n=5 each group). rh-Wnt3A treatment does not affect the density of Iba1+ microglia in ganglionic eminences (lateral ventricular wall) and corona radiate of rabbits with IVH. B) mRNA expressions of TNFα, IL1β, IL 6, and LIF was assayed in saline and rh-Wnt3A treated pups with IVH at D3 and D7. TNFα, IL-1β, and IL-6 were higher in Wnt3A treated rabbits with IVH compared to controls. IL-1β levels were decreased in Wnt3A treated rabbits with IVH relative to controls at D7. *P<0.05, **P<0.01 pups with vs. without IVH. #P<0.05, ## P<0.01 vehicle vs. rh-Wnt3A treated pups with IVH

Article Snippet: Their assay IDs were as follows: GAPDH (Oc03823402_g1), TNFα (Oc03397716_g1), IL1β (Oc03823250_s1), CNTF (Oc03397817_m1), LIF (Hs01055668_m1), IL-6 (Oc04097053_m1), Hes 5 (Mm00439311_g1), Hes 1 (APH49WG), GSk3b(Hs01047719_m1).

Techniques: Immunofluorescence, Staining, Saline

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells

doi: 10.1186/s13046-019-1056-8

Figure Lengend Snippet: The GPER antagonist G-15 reduces STAT3 nuclear accumulation triggered by estrogens. a Immunofluorescence staining of STAT3 in MDA-MB 231 cells treated for 1 h with 100 nM E2 and 100 nM G1 alone or in combination with 100 nM GPER antagonist G-15. Cells were probed with rabbit anti-STAT3 primary antibody followed by FITC-conjugated secondary antibody in order to detect STAT3 displayed by the green signal, whereas the blue signal indicates the nuclei counterstained with DAPI. Images shown are representative of 10 random fields. b Fluorescence intensities of the green signal were quantified in at least 10 random fields in each condition from three independent experiments and data are expressed as fold changes of relative fluorescence units (RFU) upon treatments respect to vehicle-treated cells. Arrows indicate STAT3 nuclear accumulation. (*) indicates p < 0.05

Article Snippet: STAT3 transcription factor signaling inhibitor STA21 and Focal Adhesion Kinase selective inhibitor VS-4718 were bought from Santa Cruz Biotechnology (DBA, Milan, Italy).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells

doi: 10.1186/s13046-019-1056-8

Figure Lengend Snippet: The FAK inhibitor VS-4718 prevents STAT3 nuclear accumulation triggered by estrogens. a Immunofluorescence staining of STAT3 in MDA-MB 231 cells treated for 1 h with 100 nM E2 and 100 nM G1 alone or in combination with 1 μM FAK kinase inhibitor VS-4718. Cells were probed with rabbit anti-STAT3 primary antibody followed by FITC-conjugated secondary antibody in order to detect STAT3 displayed by the green signal, whereas the blue signal indicates the nuclei counterstained with DAPI. Images shown are representative of 10 random fields. b Fluorescence intensities of the green signal were quantified in at least 10 random fields in each condition from three independent experiments and data are expressed as fold changes of relative fluorescence units (RFU) upon treatments respect to vehicle-treated cells. Arrows indicate STAT3 nuclear accumulation. (*) indicates p < 0.05

Article Snippet: STAT3 transcription factor signaling inhibitor STA21 and Focal Adhesion Kinase selective inhibitor VS-4718 were bought from Santa Cruz Biotechnology (DBA, Milan, Italy).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells

doi: 10.1186/s13046-019-1056-8

Figure Lengend Snippet: c-FOS, EGR1 and CTGF regulation by FAK and STAT3. c-FOS ( a ), EGR1 ( b ) and CTGF ( c ) luciferase promoter activity in MDA-MB 231 cells treated for 18 h with 100 nM E2 and 100 nM G1 alone or in combination with 100 nM GPER antagonist G15 or 20 μM STAT3 inhibitor STA21. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle were set as 1-fold induction upon which the activities induced by treatments were calculated. Each data point represents the mean ± SD of three independent experiments performed in triplicate. d c-FOS, EGR1 and CTGF mRNA expression measured by real time-PCR in MDA-MB 231 cells treated for 4 h with 100 nM E2 and 100 nM G1 alone or in combination with 100 nM GPER antagonist G15 or 20 μM STAT3 inhibitor STA21. Values normalized to the 18 s expression are shown as fold changes of the mRNA expression induced by treatments compared to cells treated with vehicle (−). e-f Immunoblots showing c-FOS, EGR1 and CTGF protein expression in MDA-MB 231 cells treated for 4 h with 100 nM E2 ( e ) and 100 nM G1 ( f ) alone or in combination with 20 μM STAT3 inhibitor STA21. Side panels show densitometric analysis of the immunoblots normalized to β-actin. g c - FOS, EGR1 and CTGF mRNA expression measured by real time-PCR in MDA-MB 231 cells treated for 4 h with 100 nM E2 and 100 nM G1 alone or in combination with 1 μM FAK kinase inhibitor VS-4718. Values normalized to the 18 s expression are shown as fold changes of the mRNA expression induced by treatments compared to cells treated with vehicle (−). Immunoblots showing c-FOS, EGR1 and CTGF protein expression in MDA-MB 231 cells treated for 4 h with 100 nM E2 ( h ) and 100 nM G1 ( i ) alone or in combination with 1 μM FAK kinase inhibitor VS-4718. Side panels show densitometric analysis of the immunoblots normalized to β-actin. Results shown are representative of three independent experiments. (*) indicates p < 0.05

Article Snippet: STAT3 transcription factor signaling inhibitor STA21 and Focal Adhesion Kinase selective inhibitor VS-4718 were bought from Santa Cruz Biotechnology (DBA, Milan, Italy).

Techniques: Luciferase, Activity Assay, Transfection, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells

doi: 10.1186/s13046-019-1056-8

Figure Lengend Snippet: The STAT3 inhibitor STA21 suppresses the migration of TNBC cells induced by E2 and G1. a Boyden Chamber assays showing the migration of MDA-MB 231 cells treated for 4 h with 100 nM E2 and 100 nM G1 alone or in combination with 20 μM STAT3 inhibitor STA21. The results are shown as cells migrating through the membrane at the bottom of the well upon treatments respect to vehicle (−). Results shown are representative of three independent experiments. b Cell migration was evaluated by wound-healing assay in MDA-MB 231 cells treated for 24 h with 100 nM E2 and 100 nM G1 alone or in combination with 20 μM STAT3 inhibitor STA21. White dotted lines indicate the wound borders at the beginning of the assay and recorded 24 h post- scratching. Results shown are representative of three independent experiments. (*) indicates p < 0.05

Article Snippet: STAT3 transcription factor signaling inhibitor STA21 and Focal Adhesion Kinase selective inhibitor VS-4718 were bought from Santa Cruz Biotechnology (DBA, Milan, Italy).

Techniques: Migration, Membrane, Wound Healing Assay