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9 protocols using sox17

1

Quantitative Western Analysis of Cell Markers

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Cell pellets from three independent experiments were lysed and western analysis performed with the LICOR quantitative fluorescence system as described (Lengfeld et al., 2017 (link)), with antibodies against human CLDN-5, OCLN, β-ACTIN (Invitrogen), and SOX17 (Abcam). Paraffin-embedded brain tissues were used to assess SOX17 expression as described (Lengfeld et al., 2017 (link)).
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2

Pluripotency and Lineage Marker Profiling

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Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT), followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min at RT. Cells were blocked for 30 min in 3% BSA in PBS and incubated with primary antibodies overnight at 4 °C. Cells were treated with fluorescently coupled secondary antibodies and were incubated for 1 h at RT. The nuclei were stained with DAPI (Sigma/merck, Billerica, MA, USA) for 5 min at RT. All images were captured using an inverted fluorescence microscope. The antibodies used in this study are summarized as follows: Oct4 (Cell Signaling Technology, Danvers, MA, USA, Cat. No. 2840, 1 : 400), Nanog (Cell Signaling Technology, Cat. No. 3580, 1 : 800), Sox2 (Epitomics, Burlingame, CA, USA, Cat. No. 2683, 1 : 500), SSEA4 (Cell Signaling Technology, Cat. No. 4755, 1 : 500), TRA-1-60 (Cell Signaling Technology, Cat. No. 4746, 1 : 1000), TRA-1-81 (Cell Signaling Technology, Cat. No. 4745, 1 : 1000), CDH1 (Cell Signaling Technology, Cat. No. 3195, 1 : 200), CDH2 (Cell Signaling Technology, Cat. No. 14215, 1 : 200), cTnT (Abcam, Cambridge, MA, USA, Cat. No. ab8295, 1 : 200), Nestin (Millipore, Cat. No. MAB5326, 1 : 1000), sox17(Abcam, Cat. No. ab155402, 1 : 200), anti-rabbit IgG, Alexa Fluor 488 (Invitrogen, Cat. No. A-11008, 1 : 1000), anti-mouse IgG, Alexa Fluor 488 (Invitrogen, Cat. No. A-11005, 1 : 1000).
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3

Quantitative Western Analysis of Cell Markers

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Cell pellets from three independent experiments were lysed and western analysis performed with the LICOR quantitative fluorescence system as described (Lengfeld et al., 2017 (link)), with antibodies against human CLDN-5, OCLN, β-ACTIN (Invitrogen), and SOX17 (Abcam). Paraffin-embedded brain tissues were used to assess SOX17 expression as described (Lengfeld et al., 2017 (link)).
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4

Quantitative Capillary Western Blot Analysis

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Cells were harvested by scraping, pelleted, washed with PBS, flash frozen and stored at −20 °C until processed. Cell pellets were resuspended in RIPA buffer (Thermo Fisher Scientific) supplemented with halt protease inhibitor cocktail (Thermo Fisher Scientific) and lysed by sonication. Lysates were cleared of debris by centrifugation at 14,000g for 15 min and quantified using the BCA protein assay kit (Thermo Fisher Scientific). Lysates were diluted 1:4 with 1X sample buffer (ProteinSimple). Protein quantification was performed using the 12–230 kDa 13 25-lane plate (PS-MK15; ProteinSimple) in a Wes Capillary Western Blot analyzer according to the manufacturer’s recommendation. Protein quantification was done using the Compass software. Primary antibodies used are as follows; OCT4 (Santa Cruz, Sc9801, 1:50), Brachyury (Cell Signaling Technology, 81694, 1:50), SOX17 (Abcam, 84990, 1:50), PAX6 (Biolegend, 901301, 1:50), TNNI3 (R&D Systems, MAB8594, 1:50), NKX2.5 (Sigma, SAB1408911, 1:50), GAPDH (Santa Cruz, sc25778, 1:2000) and tubulin (Novus, NB600–936SS, 1:50).
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5

Immunohistochemical Profiling of Molecular Markers

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IHC staining was performed as previously described [29 (link)]. Antibodies included: SOX17 (abcam, Cambridge, UK), MAML3 (abcam), β-catenin (abcam), cyclinD1 (abcam), c-Myc (abcam) and Ki67 (abcam). Scoring was performed by two independent pathologists blinded to clinicopathologic data. Protein staining was evaluated as described [30 (link)].
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6

Immunolabeling of Pluripotency and Lineage Markers

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For immunolabeling, cells were fixed in prechilled PBS with 4% paraformaldehyde for 15 min and permeabilized in PBS with 0.25% Triton X-100 for 10 min at room temperature. Nonspecific binding sites were blocked for 1 h by PBS containing 1% bovine serum albumin and 0.1% Tween 20. The fixed cells were incubated overnight at 4 °C with antibodies specific for OCT4 (5 μg/ml, rabbit polyclonal, Abcam, Nanchang, China), SSEA-4 (15 μg/ml, mouse monoclonal, Abcam), Nanog (1:200, rabbit monoclonal, Abcam), E-cadherin (1:100, mouse monoclonal, Abcam), Sox17 (1:50, mouse monoclonal, Abcam), FOXA2 (1:350, rabbit monoclonal, Abcam), AFP (5 μg/ml, mouse monoclonal, Abcam), ALB (1:500, rabbit monoclonal, Abcam), and AAT (1:50, rabbit monoclonal, Abcam). Specific labeling was visualized using secondary donkey anti-mouse or anti-rabbit antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 568 (Jackson, Nanchang, China). Nuclei were visualized by staining with DAPI (Thermo Fisher).
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7

Immunoprecipitation of SOX17, MAML3, and c-Myc

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Immunoprecipitation was performed as previously described [32 (link)]. Protein A+G Agarose beads (40μl per reaction) were added (Beyotime, Jiangsu, China). Immunoprecipitated proteins were then subjected to SDS-PAGE and western blot analysis. Antibodies used for IP were as follows: SOX17 (abcam), MAML3 (abcam), HA (abcam) and c-Myc (abcam).
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8

CUT&RUN for Epigenetic Profiling

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CUT&RUN was performed based on the CUTANA CUT&RUN protocol v1.6 (Epicypher). Isolated nuclei from FT282 and KURAMOCHI were bound to Concalavin A beads and incubated overnight at 4°C with primary antibody for H3K27me3 (Cell Signaling, RRID:AB_2798370), H3K27ac (Diagenode, RRID:AB_2637079), MECOM (Cell Signaling, AB_2184098), PAX8 (Novus, RRID:AB_2283498), SOX17 (Abcam, RRID:AB_2801385), WT1 (Santa Cruz, RRID:AB_632611). Samples were washed after overnight incubation and treated with pAG-Mnase for 10 minutes at room temperature followed by activation by calcium chloride for 2 hours at 4°C. The reaction was quenched with STOP buffer and DNA was extracted with a DNA purification kit (Epicypher). Library preparation was conducted with the NEBnext Ultra II kit with the following protocol adjustments. The second incubation during the end prep was extended to 1 hour at 50 degrees and all SPRI select steps were conducted at 1.5x volume. Cycling parameters were set to parameters as instructed in the CUT&RUN protocol v1.6 (Epicypher). Processing and peak calls were made using the same pipeline as the CUT&TAG pipeline.
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9

Differentiation of Pluripotent Stem Cells

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Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM-F12), penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Gibco. Anti-human CD34-PE, CD44-FITC, CD45-FITC, CD73-PE, CD90-FITC, and HLA-DR-FITC were obtained from BD Biosciences. Adipogenic induction medium and osteogenic induction medium Cyagen. Primary antibodies (Sox17, Cxcr4, Pdx1, and Glucagon) and fluorescent secondary antibodies were purchased from Abcam. Primary antibodies (Nkx6.1, Insulin, Somatostatin) were purchased from CST. A83-01 and SB203580 were purchased from Tocris. LDE225 were obtained from Selleck. Activin, Noggin human and other small molecule compounds were purchased from Sigma. Matrigel were purchased from Corning.
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