Journal: Journal of Endocrinology

Article Title: Cellular insulin resistance disrupts hypothalamic mHypoA-POMC/GFP neuronal signaling pathways

doi: 10.1530/joe-13-0334

Figure Lengend Snippet: Figure 5 Insulin pretreatment attenuates insulin signal transduction in mHypoA- POMC/GFP-1 neurons. Cells were pretreated with either 100 nM insulin (C) or PBS (K) for 24 h, washed with PBS for 1 h, and then re-challenged with 10 nM insulin or PBS for 15 min. Results are normalized to levels of total protein or Gb (meansGS.E.M., nZ3–4 independent experiments). Repre- sentative blots are shown below each graph. Insulin pretreatment severely attenuates insulin-induced phosphorylation of AKT (***P!0.001 by one- way ANOVA) and of FOXO1 in mHypoA-POMC/GFP-1 (*P!0.05). Total IRb levels are also reduced by insulin pretreatment in mHypoA-POMC/GFP-1 (***P!0.001).

Article Snippet: The anti-Akt, phospho-specific-Akt (Ser473), ERK1/2 (p44/p42), phospho-specific-ERK1/2 (Thr202/Tyr204), IRb, antiFOXO1, and phospho-specific-FOXO1 (Ser256) antibodies were obtained from Cell Signaling Technology, Inc.

Techniques: Transduction, Phospho-proteomics

Journal: Cell reports

Article Title: Hepatic palmitoyl-proteomes and acyl-protein thioesterase protein proximity networks link lipid modification and mitochondria

doi: 10.1016/j.celrep.2023.113389

Figure Lengend Snippet:

Article Snippet: Phospho-FoxO1 (Ser256) Rabbit mAb , Cell Signaling Technology , Cat# 84192; RRID:AB_2800035.

Techniques: Virus, Recombinant, Staining, Bicinchoninic Acid Protein Assay, Reverse Transcription, Glucose Assay, Colorimetric Assay, Mass Spectrometry, Plasmid Preparation, Software, Transfection, Blocking Assay, Protease Inhibitor

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: Generation of FoxO1-S253A/A mice and postprandial hyperglycemia. (A) The genomic locus of FoxO1 in mouse genome. (B) Gene-targeting strategy for generation of FoxO1-S253A mutation via embryonic stem (ES) cell and homologous recombination (HR). (C and D) Southern blot was performed for screening the 5′- and 3′-end HR in FoxO1 genomic loci. DNA sequencing of endogenous FoxO1 loci of negative and positive ES cells. (E) Body weight curve of mice of control wild-type (WT), heterozygous (A/+), and homozygous (A/A) mice at the ages of 4 to 16 weeks. (F) Effect of insulin on FoxO1 protein and phosphorylation (p) in hepatocytes. Primary hepatocytes were isolated from the control and A/A mice and treated with 100 nM insulin for 30 minutes, and 100 µg protein of cell lysates was subjected to Western blot against the antibody of FoxO1, T24, S316, and β-actin. (G and H) Blood glucose levels were measured in WT, A/+, and A/A mice at the ages 4 to 16 weeks during (G) 16 hours fasting and (H) random-fed conditions. *P < 0.05 vs WT and A/+ mice, n = 10 mice per group. M, Marker; Neo, neomycin; PGK-1, phosphoglycerate kinase 1; TK, thymidine kinase; utr, untranslated region.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: Mutagenesis, Homologous Recombination, Southern Blot, DNA Sequencing, Control, Phospho-proteomics, Isolation, Western Blot, Marker

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: FoxO1-S253A/A mice exhibit impaired insulin and glucagon secretion and glucose homeostasis. (A) GIR of control and A/A mice at the ages of 16 weeks in hyperinsulinemic-euglycemic clamp assays; n = 8 mice per group. (B) HGP of mice during the clamp assay. (C and D) Glucose uptake in a variety of tissues of mice during the clamp assay. P < 0.05, n = 8 mice per group. (E) GTTs were performed in 10-week mice (n = 6) under a 16-hour fasting condition. Blood glucose was plotted against the time after the IP 2-g d-glucose/kg body weight glucose injection. (F) Serum insulin concentration was measured in mice during 16-hour fasting and random-fed states. *P < 0.05 vs WT mice, n = 10 mice per group. (G) Serum insulin concentration was measured in mice during the GTT from (E). *P < 0.05 vs WT mice, n = 10 mice per group. (H) PTTs were conducted in unfed mice after 16 hours overnight. Blood glucose levels (means ± SEM, n = 6 mice per group) were determined at the indicated time points after an IP injection of 2 g pyruvate/kg body weight. *P < 0.05 vs WT. (I) Serum glucagon concentration was measured in mice during the 16-hour fasting and random-fed states. *P < 0.05 vs WT mice, n = 10 mice per group. (J) Expression of Pck1 and G6pc in the liver of WT, A/+, and A/A under a 16-hour fasting or random-fed condition. *P < 0.05 vs WT, n = 6. Brown.ad, brown adipose; Gastro, gastrocnemius; Vastus L., vastus lateralis.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: Control, Injection, Concentration Assay, Expressing

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: EE increases in FoxO1-S253A/A mice. The mice at the age of 16 weeks old were placed in metabolic cages for the measurement of food intake, physical activity, and VO2, and EE. (A) Food intake, (B) physical activity, (C) VO2, (D) EE, and (E) RER were calculated. #P < 0.05 vs WT, n = 8 mice per group.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: Activity Assay

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: FoxO1-S253A/A mice exhibit impaired α-cells, insulin, and glucagon synthesis in the pancreas. (A) Representative pancreas of control (WT) and A/A mice at the age of 12 weeks at random-fed states. *P < 0.05 vs WT, n = 6. (B and C) Insulin and glucagon concentration was measured in the pancreas of control and A/A mice at the age of 12 weeks at random-fed states. *P < 0.05 vs WT, n = 3. (D and E) Insulin and glucagon concentration was measured in the islets of control and A/A mice and normalized by islet protein. *P < 0.05 vs WT, n = 3. (F) Insulin secretion from the islets isolated from control and A/A mice. *P < 0.05 vs WT, n = 3. (G) Glucagon secretion from the islets isolated from control and A/A mice under 3.3 mM glucose condition. *P < 0.05 vs WT, n = 3. (H) Immunostaining of pancreas from 16-week-old random-fed mice by glucagon and insulin. Representative images are shown. *P < 0.05 vs WT, n = 6 mice per group. (I–K) The number of (I) α-cells and (J) β-cells was counted from at least three mice per group, and the (K) ratio of α-cell/β-cell number in each islet was counted and calculated. *P < 0.05 vs WT, n = 3 mice per group. (L) The representative images of the pancreas and islets of WT and A/A mice. (M) The area of islets in total pancreas was calculated in WT and A/A mice. *P < 0.05 vs WT, n = 4. (N and O) The expression of marker genes responsible for the pancreatic β-cell, α-cell, acinar cell, and progenitor cell in (N) total pancreas or (O) islets isolated from WT and A/A mice. *P < 0.05 vs WT, n = 3. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: Control, Concentration Assay, Isolation, Immunostaining, Expressing, Marker

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: FoxO1-S253A/A mice exhibit impaired glucagon tolerance test and hepatic gene expression. (A) Glycogen concentration in the liver of mice at 16-hour fasting and random-fed states. *P < 0.05 vs control, n = 6 mice per group. (B) Blood glucose in random-fed mice injected by glucagon. *P < 0.05 vs control, n = 8 per group. (C) Glucagon tolerance tests in control and A/A mice at the age of 12 weeks. Blood glucose was measured in 18-hour unfed mice by 16 µg/kg body weight of glucagon IP injection. *P < 0.05 vs control, n = 8 mice per group. (D) HGP assay in primary hepatocytes of control and A/A mice. Cells were stimulated by 100 nM glucagon, and glucose concentrations in culture medium were measured. *P < 0.05 WT vs WT + glucagon; #P < 0.05 S253A/A vs S253A/A + glucagon, n = 3 experiments. (E) HGP, glycogenolysis, and gluconeogenesis were measured in the primary hepatocytes. *P < 0.05 vs WT; #P < 0.05 vs A/A, n = 3 experiments. (F) Gene-expression levels of G6pc were measured in primary hepatocytes by real-time PCR. Data presented as average of triplicate determinations from control and A/A or 100 nM glucagon stimulation for 8 hours. *P < 0.05, n = 3. PCR primer sequences are cyclophilin 5′-ctaaagcatacaggtcctggcatcttg-3′ and 5′-tgccatccagccattcagtcttg-3′; G6pc are 5′-cattgtggcttccttggtcc-3′ and 5′-ggcagtatgggataagactg-3′. (G) Nuclear (N) and cytoplasmic (C) proteins were extracted from control and A/A primary hepatocytes, with or without 100 nM glucagon (Gcg) stimulation for 30 minutes. Cytoplasmic proteins (100 µg) and 20 µg nuclear proteins were subjected to SDS-PAGE and Western blot analysis. Quantification of nuclear FoxO1 was normalized by histone-H1 with analyses from ImageJ. *P < 0.05 vs control, n = 3 experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; N.S., no significance.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: Gene Expression, Concentration Assay, Control, Injection, Real-time Polymerase Chain Reaction, SDS Page, Western Blot

Journal: Endocrinology

Article Title: Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice

doi: 10.1210/en.2018-00853

Figure Lengend Snippet: Dephosphorylation of FoxO1-S253 in FoxO1-S253A/A mice regulates hepatic gluconeogenesis, glycogen metabolism, and pancreatic plasticity. (A) FoxO1-S253 de-phosphorylation controls insulin and glucagon production in the pancreas, regulating liver glucose metabolism in the feeding state, resulting in an increase of blood glucose. (B) At the fasting state, where blood glucagon decreases, reducing the blood glucose. →, stimulation; ˧, inhibition.

Article Snippet: Antibodies against FoxO1 [catalog no. 2880; Cell Signaling Technology ( 16 ), Danvers, MA], phosphorylated FoxO1-Thr24 [catalog no. 2599; Cell Signaling Technology ( 17 )], phosphorylated FoxO1-S253 [catalog no. 9461; Cell Signaling Technology ( 18 )], phosphorylated FoxO1-Ser316 [catalog no. 2486; Cell Signaling Technology ( 19 )], glyceraldehyde 3-phosphate dehydrogenase [catalog no. 2118; Cell Signaling Technology ( 20 )], β -actin [catalog no. 4970; Cell Signaling Technology ( 21 )], and histone H1 antibody [catalog no. sc-8030; Santa Cruz Biotechnology ( 22 ), Dallas, TX] were used for Western blot.

Techniques: De-Phosphorylation Assay, Inhibition

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: MST1 regulates nuclear localization of FOXO1 at tip ECs. a Images of CD31 + retinal vessels and distribution of FOXO1 of whole retina in WT mouse at P6. The red dashed lines separate into tip ECs, vascular front and vascular plexus from top to bottom. Scale bars, 200 μm. b Magnified images of CD31 + vessels and subcellular localization of FOXO1 at indicated portions. Scale bars, 50 μm. c Magnified images of the nuclear localization of FOXO1 (yellow arrowheads) at tip ECs in WT and Mst1 i∆EC mice. Scale bars, 50 μm. d Images of angiopoietin-2 (Angpt2) expression and CD31 + vessels at vascular front in WT and Mst1 i∆EC mice. Scale bars, 100 μm. e Comparisons of indicated parameters in WT ( n = 5) and Mst1 i∆EC ( n = 5) mice. Data represent mean (bar) ± s.d. (error bars). P values, versus WT by two-tailed unpaired t -test. NS not significant. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: FOXO1 is required for establishing endothelial polarization. a Diagram depicting the experiment schedule for EC-specific deletion of FOXO1 in retinal vessels from P1 and their analyses at P6. b , c Images of CD31 + vessels and comparisons of indicated parameters in WT ( n = 5) and Foxo1 i∆EC ( n = 5) mice. Scale bar, 500 μm. d Images showing VECAD and ERG + nuclei of ECs at the vascular front of WT and Foxo1 i∆EC mice. Middle and bottom panels show VE-cadherin (VECAD) and ERG signals of insets (dashed-line boxes) in top panels. Scale bars, 100 μm. e Magnified images of CD31 + vessels and ERG + nuclei of ECs. Scale bars, 50 μm. f 3D reconstructed images of CD31 + vessels and ERG + nuclei of ECs in WT and Foxo1 i∆EC mice. g Images of CD31 + vessels, ERG + nuclei of ECs and GM130 + Golgi apparatus at tip ECs in WT and Foxo1 i∆EC mice. The yellow dashed line outlines CD31 + vessels. Note that GM130 + Golgi apparatus are polarized towards the anterior or posterior of the nuclei in tip ECs of WT mice (yellow arrowheads), while such polarization is lost in tip ECs of Foxo1 i∆EC mice (yellow arrows). Scale bars, 20 μm. h Comparisons of indicated parameters in WT ( n = 5) and Foxo1 ∆EC ( n = 5) mice. Data represent mean (bar) ± s.d. (error bars). p values, versus WT by two-tailed unpaired t -test. NS not significant. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: Hypoxia activates the MST1–FOXO1 cascade in primary cultured ECs. a GSEA analyses of the microarray data (GSE19284) obtained from isolated tip ECs and non-ECs by laser capture microdissection. b , c Immunoblot analyses and temporal changes of indicated proteins in HUVECs exposed to hypoxia (1% O 2 ) for indicated times ( n = 3, each group). Center line, median; Box limits, upper and lower quartiles; Whiskers, s.d. p values versus 0 h by one-way ANOVA with Tukey’s post hoc test. NS not significant. d Immunoblot analyses of indicated proteins in HUVECs under normoxia (−) and hypoxia (+) in absence (−) or presence (+) of Trolox treatment. e Immunoblot analyses of indicated proteins in HUVECs under normoxia (−) and hypoxia (+) in absence (−) or presence (+) of Rotenone treatment. f Immunoprecipitation analysis in HUVECs with control anti-IgG and anti-FOXO1 antibody followed by immunoblotting with anti-MST1 antibody. g Immunoblot analyses of indicated proteins in siCont-ECs and siMST1-ECs under hypoxia. h Schematic picture depicting a hypoxia-intracellular ROS–MST1–FOXO1 cascade and the conserved phosphorylation site of FOXO1 by MST1. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Cell Culture, Microarray, Isolation, Laser Capture Microdissection, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: MST1 activation governs to promote nuclear import of FOXO1 under hypoxia. a – c Images and comparisons of the nuclear enrichment of FOXO1 in siCont-ECs and siMST1-ECs exposed to normoxia or hypoxia (1% O 2 ) in the absence (−) or presence (+) of VEGF (200 ng/ml) for 30 min ( n = 3, each group). Scale bars, 20 μm. Data represent mean (bar) ± s.d. (error bars). P values, normoxia with VEGF versus hypoxia with VEGF by one-way ANOVA with Tukey’s post hoc test. NS not significant. d Immunoblot analyses of indicated proteins in nuclear and cytoplasmic fractions of HUVECs exposed to normoxia (−) or hypoxia (1% O 2 ) (+) without (−) or with (+ ) VEGF stimulation (200 ng/ml, 30 min). e Images and comparisons of the nuclear enrichment of GFP in HEK293T cells transfected with gene constructs encoding either GFP-tagged FOXO1 (FOXO1-WT or WT) or non-phosphorylatable FOXO1 (FOXO1-S212A or S212A) together with either control vector (CTL) or gene construct encoding MST1 (FLAG-MST1 or MST1) [ n = 161(CTL/WT), 164(MST1/WT), 151(CTL/S212A), 154(MST1/S212A)]. Scale bars, 10 μm. Data represent mean (bar) ± s.d. (error bars). P values, CTL/WT versus MST1/WT or CTL/S212A versus MST1/S212A by one-way ANOVA with Tukey’s post hoc test. NS not significant. f Images of subcellular localizations of FOXO1 in CD31 + retinal vessels of WT and Mst1 i∆EC mice at P6. Note that the nuclear enriched FOXO1 at tip ECs (yellow arrowheads) is impaired in Mst1 i∆EC mice, while the distributions of FOXO1 at vascular front and plexus are unaltered. Scale bars, 50 μm. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Activation Assay, Western Blot, Transfection, Construct, Plasmid Preparation

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: MST1–FOXO1 cascade establishes cell polarity in EC migration. a Images of phalloidin + actin cytoskeleton and caveolin in indicated ECs subjected to the wound scratch. The dashed lines indicate the initial margin of wound scratch. Scale bars, 200 μm. b Schematic pictures depicting cell morphology at the leading edge in indicated ECs. c Comparisons of indicated parameters in indicated ECs. n = 15, each group in the left panel. n = 10–15, each group at each time in the right panel. d Comparisons of indicated parameters in indicated ECs. n = 1941(siCont), 2190(siMST1), 2217(siFOXO1) in the left panel. n = 24–25, each group in the right panel. e Polar plots showing net displacement. n = 94(siCont), 98(siMST1), 88(siFOXO1). f Schematic picture depicting net displacement and total migrating path and comparison of single cell directional persistence defined by net displacement divided by total migrating path length in indicated ECs. n = 47(siCont), 61(siMST1), 52(siFOXO1). g Images of phalloidin + actin cytoskeleton, α-tubulin + microtubule, GM130 + Golgi apparatus and DAPI in the leading edge of indicated ECs at 9 h after initiating cell migration. Note that Golgi apparatus and microtubule (red and yellow arrows) in siCont-ECs are localized in the direction of cell migration, while those (red and yellow arrowheads) in siMST1-ECs and siFOXO1-ECs are localized randomly. Moreover, siMST1-ECs and siFOXO1-ECs rarely show lamellipodia (white arrowheads) compared to siCont-ECs (white arrows). Scale bars, 50 μm. h Polar plots showing Golgi apparatus [ n = 36(siCont), 41(siMST1), 38(siFOXO1)] and microtubule organizing centre (MTOC) polarization [ n = 45(siCont), 57(siMST1), 41(siFOXO1)]. i Schematic picture summarizing predominant role of MST1 in the nuclear import of FOXO1 against VEGF/VEGFR2-PI3K/AKT pathway at tip ECs. c , d , f Box plots represent Center line, median; Box limits, upper and lower quartiles; whiskers, s.d. Right panel in c represents mean (points) ± s.d. (error bars). Right panel in d represents Bar, mean; Points, median. P values, versus siCont by one-way ANOVA with Tukey’s post hoc test. NS not significant. e , h the bold lines indicate 120° region centered on the vector which is vertical to the wound scratch direction. The numbers indicate the frequency of dots within the 120° region of the bold line. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Migration, Plasmid Preparation

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: MST1–FOXO1 cascade is required for pathologic angiogenesis. a Images of CD31 + vessels in the superficial layer of retinas and avascular area (red) in WT-OIR, Mst1 i∆EC -OIR, and Foxo1 i∆EC -OIR mice. Scale bars, 500 μm. b Images of subcellular localization of FOXO1 in CD31 + vessels at vascular front (revascularization) and vascular plexus (neovascularization) in WT-OIR, Mst1 i∆EC -OIR, and Foxo1 i∆EC -OIR mice. Scale bars, 100 μm. Note that WT-OIR mice exhibited a nuclear localization of FOXO1 (yellow arrowheads), while Mst1 i∆EC -OIR mice showed a diffuse nucleocytoplasmic localization of FOXO1 (yellow arrows) in tip ECs and NVT ECs. c Comparisons of indicated parameters in WT-OIR ( n = 5), Mst1 i∆EC -OIR ( n = 5) and Foxo1 i∆EC -OIR ( n = 5) mice. Data represent mean (bar) ± s.d. (error bars). P values, versus WT by two-tailed unpaired t -test. d Images of CD31 + vessels, ERG + nuclei of ECs and GM130 + Golgi apparatus at tip ECs in WT-OIR, Mst1 i∆EC -OIR and Foxo1 i∆EC -OIR mice. The images of the inset (white dashed-line boxed) are magnified in e . The yellow dashed line outlines CD31 + vessels. Scale bars, 50 μm. e Images of ERG + nuclei of ECs and GM130 + Golgi apparatus at tip ECs in WT-OIR, Mst1 i∆EC -OIR, and Foxo1 i∆EC -OIR mice. The yellow dashed line outlines CD31 + vessels. Note that GM130 + Golgi apparatus are polarized towards the anterior or posterior of the nuclei in tip ECs of WT-OIR mice (yellow arrowheads), while such polarization is lost in tip ECs of Mst1 i∆EC -OIR and Foxo1 i∆EC -OIR mice (yellow arrows). Scale bars, 100 μm. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Two Tailed Test