Journal: Molecular oncology

Article Title: SETD2 loss in renal epithelial cells drives epithelial-to-mesenchymal transition in a TGF-β-independent manner.

doi: 10.1002/1878-0261.13487

Figure Lengend Snippet: Fig. 7. SOX2, OCT2, and PRRX1 are downstream effectors of the SETD2-regulated EMT program. (A) Expression of SOX2, OCT2, and PRRX1 in TGF-b-treated WT (72 h), SETD2 KO, and SETD2 rescue tested by RT-qPCR (run in triplicate). (B) Migration capacity by wound healing assay, (C) invasiveness by transwell assay, and (D) stemness by 3D spheroid formation assay in RPTEC WT GFP (control vector), SETD2 KO1 and KO2, and SOX2/OCT2/PRRX1-transduced WT RPTEC lines. Images are taken at 49 magnification, scale bar: 1000 lm for (B) and (D) and at 2.59 magnification, scale bar: 1200 lm for (C). Data are represented as mean SEM for triplicate reactions for B–D. P-value is calculated by one-way ANOVA in (A), (B), and (D). Two-way ANOVA is used for statistical test for (C). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, P ≥0.05. (E) Model of the SETD2 loss-driven EMT program through cell intrinsic (transcriptional) and cell extrinsic (paracrine) mechanisms.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense #ab1791), SOX2 (R&D Systems; #AF2018-SP), OCT2 (Thermo Fisher; #39-5400), PRRX1 (Novus Biologicals, St. Charles, MO, USA; #NBP1-06067), anti-FLAG (Sigma-Aldrich; #F1804), GAPDH (Cell Signaling Technology; #2118S), and lamin B1 (Proteintech; #12987-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Migration, Wound Healing Assay, Transwell Assay, Tube Formation Assay, Control, Plasmid Preparation

Journal: Nature Communications

Article Title: NR6A1 is essential for neural crest cell specification, formation and survival

doi: 10.1038/s41467-026-68647-2

Figure Lengend Snippet: A In situ hybridization of neuroepithelial markers (purple stain) in null and wildtype littermate control embryos at E9.0. Sox2 , Wnt1 and Pax3 are all expressed in the Nr6a1 null embryos, indicating the neural ectoderm has formed and patterned. B Immunostaining of SOX2 (green), PAX3 (green) and pHH3 (red) on transverse histological sections at E9.0. SOX2 and PAX3 are expressed in the neural plate in both null and control embryos. SOX2 expression is expanded to dorsal regions of the neural plate. PAX3 expression is maintained in the Nr6a1 null, although no PAX3 positive migratory cells could be observed further demonstrating the NCC deficiency. More pHH3 staining can be seen in the neural plate of the Nr6a1 null embryos suggesting the neural ectoderm is maintained in a highly proliferative state. A minimum of 5 embryos were assayed for each in situ hybridization marker and cell proliferation assay.

Article Snippet: The monoclonal antibodies SOX2 (R & D Systems, Minneapolis, MN) and pHH3 (Upstate/Millipore, Billerica, MA) were both used at 1:500.

Techniques: In Situ Hybridization, Staining, Control, Immunostaining, Expressing, Marker, Proliferation Assay

Journal: Stem Cells Translational Medicine

Article Title: Genome Editing in Neuroepithelial Stem Cells to Generate Human Neurons with High Adenosine‐Releasing Capacity

doi: 10.1002/sctm.16-0272

Figure Lengend Snippet: Generation of ADK‐deficient neural stem cells. (A): Long‐term self‐renewing neuroepithelial stem cells (lt‐NES cells) stably proliferate in the presence of the growth factors FGF2 and EGF and show rosette‐like morphology with expression of NESTIN, PLZF, and SOX2 as well as polarized expression of the tight junction marker ZO‐1. Following growth factor withdrawal, lt‐NES cells differentiate into a major fraction of beta III‐tubulin‐ and MAP2ab‐positive neurons and a minor fraction of GFAP‐positive astrocytes. (B): Quantitative PCR analysis of adenosine kinase (ADK) in lt‐NES cells and their differentiated progeny, normalized to RPLP0 as housekeeping gene and compared to ADK expression in human fetal brain RNA. (C): Cartoon demonstrating the zinc finger nuclease gene disruption strategy in exon 5 of the ADK gene. A pair of zinc finger nucleases induces a targeted double strand break (red arrows) after binding to their target site (indicated by blue lines). Repair via nonhomologous‐end‐joining can drive a frame shift resulting in premature disruption of translation (blue arrow). The catalytic core around Asp316 is situated in exon 10 (of 11) at the C‐terminal end of the protein. Introduction of frame shifts in front of the catalytic part leads to elimination of all enzymatic activity. (D): Sequencing of the target locus revealed a homozygous deletion of 1 bp (thymidine 165 of exon 5) in 81.7% of the 148 analyzed clones (red arrow). (E): This frame shift leads to a stop codon (asterisk) after 161 translated amino acids, resulting in a complete loss of ADK activity. Scale bars: 50 µm. Abbreviations: ADK, adenosine kinase; EGF, epidermal growth factor; FGF2, basic fibroblast growth factor; GFAP, glial fibrillary acidic protein; lt‐NES cell, long‐term self‐renewing neuroepithelial stem cell; PCR, polymerase chain reaction.

Article Snippet: Primary antibodies (beta III‐tubulin: Biolegend (San Diego, CA) PRB‐435P, 1:1,000; DACH1: Proteintech (Rosemont, IL) 10914‐1‐AP, 1:500; glial fibrillary acidic protein (GFAP): Agilent (Santa Clara, CA) ZO334, 1:500; MAP2ab: Sigma‐Aldrich M1406, 1:1,000; Nestin: R&D Systems MAB1259, 1:500; PLZF: EMD Millipore (Billerica, MA) OP128, 1:50; Sox2: R&D Systems MAB 2018, 1:500; ZO‐1: Life Technologies 61–7300, 1:100) were applied overnight at 4°C in blocking solution.

Techniques: Stable Transfection, Expressing, Marker, Real-time Polymerase Chain Reaction, Zinc-Fingers, Disruption, Binding Assay, Activity Assay, Sequencing, Clone Assay, Polymerase Chain Reaction

Journal: Stem Cells Translational Medicine

Article Title: Genome Editing in Neuroepithelial Stem Cells to Generate Human Neurons with High Adenosine‐Releasing Capacity

doi: 10.1002/sctm.16-0272

Figure Lengend Snippet: Characterization of ADK‐deficient neural stem cells. (A): Morphology and marker expression of ADK −/− long‐term self‐renewing neuroepithelial stem cells were comparable to wild‐type cells with rosette‐like appearance and the expression of NESTIN, PLZF, SOX2, and polarized ZO‐1 expression in the proliferating stage as well as expression of beta III‐tubulin, MAP2ab and GFAP upon 5 weeks of differentiation. Scale bars: 50 µm. (B): Absence of ADK protein was confirmed by Western blot analysis. Actin served as loading control. Mouse brain protein served as positive control. (C): Quantitative analysis reveals comparable numbers of beta III‐tubulin‐, MAP2ab‐, and GFAP‐positive cells in differentiated populations derived from wild‐type and ADK −/− cells. (D): Following transplantation, both populations efficiently differentiated into neurons (detected by a human‐specific antibody to neurofilaments HO14). Human synaptophysin‐positive punctae were detected in neuronal projection regions (enlarged regions show synaptophysin‐positive dots in close vicinity to human neurofilaments marked by arrowheads). No significant difference was detected when we compared the ratio of synaptophysin‐positive punctae to the length of human neurofilament‐positive fibers, suggesting that wild‐type and ADK −/− cells exhibit comparable in vivo differentiation and integration potential (unpaired t test, p = .6705; scale bars: 50 µm). (E): Relative viability of WT or ADK −/− cells after 48 hours exposure to increasing concentrations of 8‐Cl‐adenosine as assessed by PrestoBlue assay. Only ADK‐deficient cells tolerated the additional chemoselection with 8‐Cl‐Adenosine, while wild‐type cells showed pronounced cytotoxicity. Abbreviations: ADK, adenosine kinase; GFAP, glial fibrillary acidic protein; WT, wild‐type.

Article Snippet: Primary antibodies (beta III‐tubulin: Biolegend (San Diego, CA) PRB‐435P, 1:1,000; DACH1: Proteintech (Rosemont, IL) 10914‐1‐AP, 1:500; glial fibrillary acidic protein (GFAP): Agilent (Santa Clara, CA) ZO334, 1:500; MAP2ab: Sigma‐Aldrich M1406, 1:1,000; Nestin: R&D Systems MAB1259, 1:500; PLZF: EMD Millipore (Billerica, MA) OP128, 1:50; Sox2: R&D Systems MAB 2018, 1:500; ZO‐1: Life Technologies 61–7300, 1:100) were applied overnight at 4°C in blocking solution.

Techniques: Marker, Expressing, Western Blot, Control, Positive Control, Derivative Assay, Transplantation Assay, In Vivo, Prestoblue Assay

Journal: Cell Cycle

Article Title: Mesenchymal traits are selected along with stem features in breast cancer cells grown as mammospheres

doi: 10.4161/cc.22543

Figure Lengend Snippet: Figure 2. ALDH1 expression in the breast cancer cell lines analyzed. ( A ) ALDH1A1 and ( B ) ALDH1A3 mRNA-relative expression determined by qRT-PCR. Data were normalized to three reference genes as described in Materials and Methods. Bars represent standard deviation. ( C ) SOX2 transcript determined by RT-PCR. GAPDH is reported as RNA control. NT2D1, NTera D1 cell line was used as a positive control for SOX2.

Article Snippet: Western blotting was performed on cell protein lysates (50 mM Tris, pH 7.6, 250 mM NaCl, 0,2% Triton X-100, 0,3% NP40, 2 nM EGTA, 2 nM EDTA, 0.1% P-40, complete protease inhibitor cocktail Roche Molecular Biochemicals) using mouse antibodies reacting to human SOX2 (R&D Systems), actin (Santacruz) and rabbit antibodies to human SNAI1 (Cell Signaling) and β-tubulin (Santacruz).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Control, Positive Control

Journal: Cell Cycle

Article Title: Mesenchymal traits are selected along with stem features in breast cancer cells grown as mammospheres

doi: 10.4161/cc.22543

Figure Lengend Snippet: Figure 3. Stem properties were modulated by MS-proficient conditions. ( A ) ALDH1A1 and ( B ) ALDH1A3 transcript levels in cells grown as primary (M1), secondary (M2) and tertiary (M3) mammospheres (gray columns) or in adherence (ctr, black columns) in the same-stem medium. Data, obtained by qRT-PCR, were normalized to three reference genes as described in Materials and Methods. Bars represent standard deviation; asterisks p < 0.05 in one-way Anova analysis. ( C ) SOX2 protein levels, determined by western blotting, in MCF7 cells grown as primary (M1), secondary (M2) and tertiary (M3) mammospheres or in adherence (ctr) in the same-stem medium. NT2D1 is reported as a positive control. The increment of SOX2, determined by the ratio of SOX2 signal over tubulin signal (used as a loading control), is reported.

Article Snippet: Western blotting was performed on cell protein lysates (50 mM Tris, pH 7.6, 250 mM NaCl, 0,2% Triton X-100, 0,3% NP40, 2 nM EGTA, 2 nM EDTA, 0.1% P-40, complete protease inhibitor cocktail Roche Molecular Biochemicals) using mouse antibodies reacting to human SOX2 (R&D Systems), actin (Santacruz) and rabbit antibodies to human SNAI1 (Cell Signaling) and β-tubulin (Santacruz).

Techniques: Quantitative RT-PCR, Standard Deviation, Western Blot, Positive Control, Control