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: International Journal of Molecular Sciences

Article Title: Generation and Characterization of hiPS Lines from Three Patients Affected by Different Forms of HPDL -Related Neurological Disorders

doi: 10.3390/ijms251910614

Figure Lengend Snippet: Primary and secondary antibodies employed for immunofluorescence assay to stain stemness and differentiation markers.

Article Snippet: Rabbit anti-SOX17 (D1T8M) , 1:500 , Cell Signaling Technology, Cat #81778.

Techniques: Immunofluorescence, Staining

Journal: Development (Cambridge, England)

Article Title: Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling.

doi: 10.1242/dev.021121

Figure Lengend Snippet: Fig. 3. BMP signaling blockade changes cell fate from mesoderm to the anterior PS/endoderm. (A) The qPCR analysis on RNA isolated at the day 3 from unstimulated hES cells (vehicle: v) and β-catenin-activated cells (OHT) with or without Noggin (Nog; 250 ng/ml) or SB431542 (SB) was performed using specific primers for the genes indicated. (B) Expression of the anterior PS/endoderm markers in cells activated by β-catenin with Noggin. The ΔNβ-cateninER cells were cultured with 4OHT in the presence or absence Noggin for 3 days, and subjected to immunostaining with T, FOXA2 and SOX17. Nuclei were counterstained with DAPI. Scale bar: 100 μm. (C) Developmental potentials of β-catenin- activated cells toward definitive endoderm and cardiac lineage. The day 3 β-catenin-activated cells with or without Noggin were cultured with BMP4 and FGF2 for additional 4 days, and analyzed by RT-PCR. The transgenic cell lines derived from different ES cell lines are shown (K1βcatER derived from KhES-1 cells, K3βcatER derived from KhES-3 cells). ES, unstimulated ΔNβ-cateninER cells.

Article Snippet: Antibodies against the following proteins were used: VEGF R2/KDR (clone#89115), SOX17 and CXCR4 (clone#44717) (R&D Systems); E-cadherin, Smad2/3, GSK3β and β-catenin (BD Biosciences); N-cadherin (GC-4, Sigma); ZO-1 (Invitrogen); Nanog (ReproCELL); HNF3β, Oct3/4, Brachyury and ERα (Santa Cruz Biotechnology, Santa Cruz, CA); SSEA3 and SSEA4 (Developmental Hybridoma Bank); TRA1-60 and TRA-1-81 (Chemicon); Phospho-Smad1 (Ser463/465), Smad1, phospho-Smad2 (Ser465/467), phospho-MAPK (Thr202/Tyr204), MAPK, phospho-Akt (Ser473), Akt and phospho-GSK3α/β (Ser21/9) (Cell Signaling, Danvers, MA).

Techniques: Isolation, Expressing, Cell Culture, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Derivative Assay