Journal: Cells

Article Title: Using High-Content Screening to Generate Single-Cell Gene-Corrected Patient-Derived iPS Clones Reveals Excess Alpha-Synuclein with Familial Parkinson’s Disease Point Mutation A30P

doi: 10.3390/cells9092065

Figure Lengend Snippet: iPS cell line characterization. ( A ) Pluripotency characterization of the three single-cell isogenic lines generated in this project. Phase contrast images of pluripotent colonies, image taken using a 5× objective. Scale bar represents 100 μM. Antibody specific pluripotency marker expression of OCT4/SOX2 and NANOG/TRA-1-60. Images taken using a 25× objective, scale bar is 50 μM. ( B ) Directed differentiation to the three embryonic germ layers using antibodies specific to the ectoderm (OTX2), endoderm (SOX17) and mesoderm (BRACHYURY). Images taken using a 25× objective, scale bar is 50 μM.

Article Snippet: Primary antibodies used provided with the kit were: SOX17 (1:1000, R&D Systems, Minneapolis, MN, USA; #963121), OTX2 (1:1000, R&D Systems, Minneapolis, MN, USA; #963273) and BRACHYURY (1:1000, R&D Systems, Minneapolis, MN, USA; #963427).

Techniques: Generated, Marker, Expressing

Journal: Journal of biomedical science

Article Title: Dysregulation of SOX17/NRF2 axis confers chemoradiotherapy resistance and emerges as a novel therapeutic target in esophageal squamous cell carcinoma.

doi: 10.1186/s12929-022-00873-4

Figure Lengend Snippet: Fig. 2 SOX17 functions as a transcriptional repressor of NRF2. A Schematic of reporter plasmid construct used in luciferase reporter assay. The NFE2L2 promoter (− 2000 ~ + 10 bp) containing seven SOX17 binding sites was sub-cloned into the pGL4 basic vector. B Schematic of the hypothesis that SOX17-mediated transcriptional repression on NRF2 model. SRY sites: SOX17 binding residues. C Dual luciferase promoter reporter assay was performed to examine the effects of SOX17 wild type (SOX17-WT) or HMG box deletion (SOX17-ΔHMG) overexpression on promoter activity of NFE2L2 in ESCC cells. D Promoter map for NFE2L2 gene. Regions examined with ChIP assay are marked by red circle-backslash symbol. E ChIP-qPCR assay was performed to measure SOX17 binding ability to the promoter region of NFE2L2 in ESCC cells. Data represents mean ± s.e.m. ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Nuclear lysates were sonicated to obtain DNA fragments around 500 bp and then subjected to immunoprecipitation for 16 h at 4 °C using 4 μg anti-SOX17 antibody (R&D Systems, Minneapolis, MN, USA) or normal IgG (negative control).

Techniques: Plasmid Preparation, Construct, Luciferase, Reporter Assay, Binding Assay, Clone Assay, Over Expression, Activity Assay, ChIP-qPCR

Journal: Journal of biomedical science

Article Title: Dysregulation of SOX17/NRF2 axis confers chemoradiotherapy resistance and emerges as a novel therapeutic target in esophageal squamous cell carcinoma.

doi: 10.1186/s12929-022-00873-4

Figure Lengend Snippet: Fig. 3 SOX17 overexpression downregulated NRF2 protein expression and the mRNA level of NRF2-targeted genes. A, B Western blot showed a decreased expression level of NRF2 protein after SOX17-WT overexpression, while no change in NRF2 protein expression after SOX17-ΔHMG overexpression in KYSE510 pair cells (A) and in CE48T pair cells (B). GAPDH was used as an internal control. C and D, The mRNA expressions of NFE2L2 and NRF2-regulated genes GPX2, AKR1C1, AKR1C2, AKR1C3, NQO1, and G6PD were determined by RT-qPCR analysis after SOX17 overexpression for 72 h in KYSE510 pair cells (C) and in CE48T pair cells (D). β-actin was used as an internal control. Data represents mean ± s.e.m. ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Nuclear lysates were sonicated to obtain DNA fragments around 500 bp and then subjected to immunoprecipitation for 16 h at 4 °C using 4 μg anti-SOX17 antibody (R&D Systems, Minneapolis, MN, USA) or normal IgG (negative control).

Techniques: Over Expression, Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: Journal of biomedical science

Article Title: Dysregulation of SOX17/NRF2 axis confers chemoradiotherapy resistance and emerges as a novel therapeutic target in esophageal squamous cell carcinoma.

doi: 10.1186/s12929-022-00873-4

Figure Lengend Snippet: Fig. 4 The effects of SOX17 and/or NRF2 overexpression on cell behaviors and tumor growth. A, B Colony formation assay of KYSE510 pair cells manipulated with SOX17 and/or NRF2 expression. The colonies were stained on day 8 after seeding (A), and the colony formation ability was quantified (B). C, D Wound healing assay of KYSE510 pair cells manipulated with SOX17 and/or NRF2 expression. Cells were monitored for their ability to migrate into the wound gap. The wound gap was photographed (C) and quantified (D) at 10 h. E, F Transwell invasion assay of KYSE510 pair cells manipulated with SOX17 and/or NRF2 expression. The invaded cells were photographed (E) and quantified (F) at 20 h. G KYSE510-R cells transfected with SOX17 and/or NRF2 were subcutaneously injected into BALB/c nude mice and observed for tumor growth. H, I Tumor size (H) and tumor weight (I) were measured at the end of the experiment. Data represents mean ± s.e.m. ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Nuclear lysates were sonicated to obtain DNA fragments around 500 bp and then subjected to immunoprecipitation for 16 h at 4 °C using 4 μg anti-SOX17 antibody (R&D Systems, Minneapolis, MN, USA) or normal IgG (negative control).

Techniques: Over Expression, Colony Assay, Expressing, Staining, Wound Healing Assay, Transwell Invasion Assay, Transfection, Injection

Journal: Journal of biomedical science

Article Title: Dysregulation of SOX17/NRF2 axis confers chemoradiotherapy resistance and emerges as a novel therapeutic target in esophageal squamous cell carcinoma.

doi: 10.1186/s12929-022-00873-4

Figure Lengend Snippet: Fig. 5 ZVI@CMC nanoparticles elicited DNMT inhibition to restore the expression of SOX17 and NRF2. A, B Colony formation assay of KYSE510 pair cells treated with ZVI@CMC. The colonies were stained on day 12 after seeding (A), and the colony number was quantified (B). C Intracellular ROS level was determined by flow cytometry analysis of DCFDA fluorescence intensity after ZVI@CMC treatment for 24 h. D Immunoblotting of DNMT1, DNMT3B, SOX17, and NRF2 in ESCC cells treated with ZVI@CMC. GAPDH was used as an internal control. E Immunofluorescence staining of β-TrCP, DNMT1, NRF2, and DAPI in KYSE510 pair cells treated with ZVI@CMC. F Methylation-specific PCR (MSP) demonstrated that ZVI@CMC could reduce the methylation of SOX17 promoter. M indicates methylated PCR products, and U indicates unmethylated PCR products. G RT-qPCR analysis showed that the mRNA expressions of NRF2 downstream genes were downregulated by ZVI@CMC treatment. β-actin was used as an internal control. Data represents mean ± s.e.m. ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Nuclear lysates were sonicated to obtain DNA fragments around 500 bp and then subjected to immunoprecipitation for 16 h at 4 °C using 4 μg anti-SOX17 antibody (R&D Systems, Minneapolis, MN, USA) or normal IgG (negative control).

Techniques: Inhibition, Expressing, Colony Assay, Staining, Flow Cytometry, Fluorescence, Western Blot, Control, Immunofluorescence, Methylation, Quantitative RT-PCR

Journal: Journal of biomedical science

Article Title: Dysregulation of SOX17/NRF2 axis confers chemoradiotherapy resistance and emerges as a novel therapeutic target in esophageal squamous cell carcinoma.

doi: 10.1186/s12929-022-00873-4

Figure Lengend Snippet: Fig. 7 The model of SOX17/NRF2 transcription axis and ZVI@CMC treatment in ESCC. In the CCRT resistant cells, the promoter hypermethylation-induced low SOX17 protein expression causes dysregulation of the cytoprotective enzymes controlled by NRF2, and thus resulting in CCRT resistance (left). Interestingly, ZVI@CMC elicits DNMT inhibition to re-express SOX17 and suppress NRF2-mediated cytoprotective programs in ESCC, and thereby sensitizing cancer cells to CCRT treatment (right)

Article Snippet: Nuclear lysates were sonicated to obtain DNA fragments around 500 bp and then subjected to immunoprecipitation for 16 h at 4 °C using 4 μg anti-SOX17 antibody (R&D Systems, Minneapolis, MN, USA) or normal IgG (negative control).

Techniques: Expressing, Inhibition

Journal: Stem Cell Reports

Article Title: Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells

doi: 10.1016/j.stemcr.2018.06.003

Figure Lengend Snippet: The Two Types of Cells Are Both Pluripotent (A) The images indicated that both MCoG and DCoG cells formed colonies and expressed AP. (B) NANOG and OCT4 were expressed both in MCoG and DCoG cells after 26 passages. (C) RT-PCR analysis of expression of pluripotency genes ( OCT4 and NANOG ) and differentiation genes ( PAX6 , ectoderm; BRACHYURY , mesoderm; and AFP , endoderm) in MCoG and DCoG cells. (D) Flow cytometric analysis of pluripotency markers in MCoG and DCoG cells. The 50,000 cells analyzed express high levels of hPSC-specific cell surface markers (TRA-1-60, SSEA-4) and low levels of differentiation-specific cell surface markers (SSEA-1). (E) Embryoid bodies formed by MCoG and DCoG cells differentiated into the three germ layers: ectoderm (β-tubulin), mesoderm (α-SMA), and endoderm (SOX17 and AFP). (F) Teratomas formed by MCoG and DCoG cells in severe combined immunodeficiency mice, containing tissues that are representative of all three embryonic germ layers. (G) Normal karyotype exhibited in both MCoG and DCoG cells. See also Figure S3 .

Article Snippet: Samples were blocked with blocking buffer (5%, v/v, normal goat serum; 5%, v/v, normal donkey serum; 3%, v/v, BSA; and 0.1%, v/v, Tween 20 in PBS) for 1 hr and incubated with the following primary antibodies diluted in blocking buffer at room temperature for 2 hr or at 4°C overnight: anti-human OCT4 (2 μg/mL, sc-5279, Santa Cruz Biotechnology, USA), anti-human NANOG (9.4 μg/mL, 4903S, Cell Signaling Technology, UK), anti-human β-tubulin III (6 μg/mL, 5568, Cell Signaling Technology), anti-human α-SMA (2 μg/mL, ab5694, Abcam, UK), anti-human SOX17 (20 μg/mL, MAB1924, R&D Systems, USA), anti-E-cadherin (1/300 dilution, 3195S, Cell Signaling Technology), and anti-MKL1 (3 μg/mL, HPA030782, Sigma-Aldrich).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing