Journal: Stem Cell Research & Therapy

Article Title: Investigation of de novo mutations in a schizophrenia case-parent trio by induced pluripotent stem cell-based in vitro disease modeling: convergence of schizophrenia- and autism-related cellular phenotypes

doi: 10.1186/s13287-020-01980-5

Figure Lengend Snippet: Establishment and molecular characterization of NPC lines and mature neuronal cultures. Investigation of target genes KHSRP and LRRC7. a Changes in gene expression patterns in NPCs and neurons derived from the case-parent trio. b , c NPCs and neurons derived from hiPSCs by the hippocampal neuronal differentiation protocol were investigated by immunofluorescence staining and visualized by confocal fluorescent microscopy. Immunocytochemical staining shows Nestin/Sox2 ( b ) and Map 2/Prox1 ( c ) positivity in these established neural cell types. Scale bars = 100 μm. d , e Immunofluorescence staining for KHSRP and LRRC7 in neurons. KHSRP ( d ) shows nuclear and cytoplasmic localization, while LRRC7 ( e ) localized postsynaptically in neurons. Scale bars = 50 μm

Article Snippet: The samples were then incubated for 1 h at room temperature with antibodies anti-SOX2 (monoclonal/mouse, 1:20 dilution; MAB2018, R&D Systems, Minneapolis, USA) and anti-Nestin (polyclonal/rabbit, 1:250 dilution; ab92391, Abcam, Cambridge, UK) or for overnight at 4 °C with antibodies anti-PROX1 (polyclonal/rabbit, 1:500 dilution; ab101851, Abcam, Cambridge, UK) and anti-MAP 2 (monoclonal/mouse, 1:500 dilution; M1406, Sigma/Merck, Darmstadt, Germany or polyclonal/rabbit, 1:1000 dilution; ab5622, Millipore, MA, USA).

Techniques: Gene Expression, Derivative Assay, Immunofluorescence, Staining, Microscopy

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: Neural regeneration research

Article Title: Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons.

doi: 10.4103/1673-5374.378203

Figure Lengend Snippet: Figure 2 ｜ The modified dual SMAD pathway inhibition protocol yielded a low proportion of dopaminergic neurons. (A) Representative images of human induced pluripotent stem cells (hiPSC) with immunostaining of pluripotency markers, octamer-binding transcription factor 4 (OCT4) and stage-specific embryonic antigen-4 (SSEA4). Scale bars: 50 μm. (B) Representative images of neural precursor cells (NPC) induced from hiPSC with immunostaining of SRY-box transcription factor 2 (SOX2) and Nestin. Scale bars: 50 μm. (C) Representative images of hiPSC-derived neurons with immunostaining of microtubule- associated protein 2 (MAP2) (green) and human nuclei (HuNu) antibody (red). Scale bars: 50 μm. (D) Analysis of neuronal differentiation efficiency (number of cells expressing HuNu: n = 443 cells in dual SMAD pathway inhibition protocol). (E) Representative images of hiPSC-derived neurons with immunostaining of markers for different neuronal subtypes: tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), vesicular glutamate transporter 1 (VGLUT1) (all in green), and MAP2 (red) grown on mouse astrocytes. Scale bars: 50 μm. (F) Differentiation ratio of dopaminergic neurons, GABAergic neurons, and glutamatergic neurons were assessed based on the proportion of TH, GABA, and VGLUT1 positive cells among MAP2 positive cells, respectively. At least three independent replicates were performed of each experiment. SMAD: Suppressor of mother against decapentaplegic.

Article Snippet: Primary antibodies included: octamer-binding transcription factor 4 (OCT4), a marker used for the identification of iPSC (rabbit, 1:100, BioVision, Milpitas, CA, USA, Cat# 6765-100, RRID: AB_2936816), stage-specific embryonic antigen-4 (SSEA4), a marker used for identification of iPSC (mouse, 1:100, Invitrogen, Rockford, AL, USA, Cat# 41-4000, RRID: AB_2533506), SRY-box transcription factor 2 (SOX2), a marker used for identification of NPC (mouse, 1:500, R&D Systems, Minneapolis, MN, USA, Cat# MAB2018, RRID: AB_358009), Nestin, a marker used for identification of NPC (rabbit, 1:1000, Millipore, Darmstadt, Germany, Cat# ABD69, RRID: AB_2744681), microtubule-associated protein 2 (MAP2), a marker of dendrites (rabbit, 1:1000, Millipore, Burlington, MA, USA, Cat# AB5622, RRID: AB_91939), MAP2 (mouse, 1:1000, Millipore, Cat# AMAb91375, RRID: AB_2716657), beta III tubulin (TUJ1), a neuronal marker (mouse, 1:1000, Sigma-Aldrich, Cat# T8660, RRID: AB_477590), anti-human nuclei (HuNu), a marker of nuclei in human cells (mouse, 1:500, Millipore, Cat# MAB1281, RRID: AB_94090), anti-vesicular glutamate transporter 1 (VGLUT1), a glutamatergic neuronal marker (mouse, 1:250, Millipore, Cat# MAB5502, RRID: AB_262185), gamma-aminobutyric acid (GABA), a GABAergic neuronal marker (rabbit, 1:1000, Sigma-Aldrich, Cat# A2052, RRID: AB_477652), tyrosine hydroxylase (TH), a dopaminergic neuronal marker (rabbit, 1:1000, Millipore, Cat# AB152, RRID: AB_390204), TH (chicken, 1:500, Millipore, Cat# AB9702, RRID: AB_570923), FOXA2, a midbrain neuronal marker (rabbit, 1:500, Cell Signaling Technology, Danvers, MA, USA, Cat# 8186, RRID: AB_10891055), and paired like homeodomain 3 (PITX3), another midbrain neuronal marker (rabbit, 1:500, Millipore, Cat# AB5722, RRID: AB_91997).

Techniques: Modification, Inhibition, Immunostaining, Binding Assay, Derivative Assay, Expressing

Journal: Neural regeneration research

Article Title: Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons.

doi: 10.4103/1673-5374.378203

Figure Lengend Snippet: Figure 4 ｜ The SHH + FGF8 differentiation protocol yielded a high proportion of dopaminergic neurons, with a small fraction of neurons that expressed FOXA2, a marker of midbrain dopaminergic neurons. (A) Representative images of neural precursor cells (NPC) induced from human induced pluripotent stem cells (hiPSC) with immunostaining of Forkhead box protein A2 (FOXA2) and SRY-box transcription factor 2 (SOX2). Scale bars: 50 μm. (B) Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of microtubule- associated protein 2 (MAP2) (green) and human nuclei (HuNu) antibody (red). Scale bars: 50 μm. (C) Comparison of the differentiation efficiency of neurons in the SHH + FGF8 protocol and modified dual SMAD pathway inhibition protocol (number of cells expressing HuNu: n = 607 cells in SHH+FGF8 protocol, n = 443 cells in dual SMAD pathway inhibition protocol). (D) Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of tyrosine hydroxylase (TH) (green) and MAP2 (red). Scale bars: 50 μm. (E) The proportion of dopaminergic neurons in the SHH + FGF8 protocol and modified dual SMAD pathway inhibition protocol was determined by calculating the percentage of TH-positive cells in MAP2-positive cells. (number of cells expressing MAP2: n = 581 cells in SHH + FGF8 protocol, n = 339 cells in dual SMAD pathway inhibition protocol). (F) The white box area in the image is enlarged and displayed on the right. NPC were infected with a DAT-Cherry lentivirus and then cultured on a glial cell feeder layer to label dopaminergic neurons in the later differentiation stage. Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of TH (green) and beta III tubulin (TUJ1) (white) on day 33. Scale bars: 50 μm (left), 10 μm (right). (G) The white box area in the image is enlarged and displayed at the bottom. Immunofluorescence staining of FOXA2 (red) and TH (green). Scale bars: 20 μm (upper panels), 5 μm (lower panels). At least three independent replicates were performed for each experiment. Data are presented as mean ± SEM, ****P < 0.0001. Statistical significance was evaluated by two-tailed unpaired Student’s t-test (C, E). FGF8: Fibroblast growth factor 8; ns: not significant; SHH: Sonic Hedgehog; TH: tyrosine hydroxylase.

Article Snippet: Primary antibodies included: octamer-binding transcription factor 4 (OCT4), a marker used for the identification of iPSC (rabbit, 1:100, BioVision, Milpitas, CA, USA, Cat# 6765-100, RRID: AB_2936816), stage-specific embryonic antigen-4 (SSEA4), a marker used for identification of iPSC (mouse, 1:100, Invitrogen, Rockford, AL, USA, Cat# 41-4000, RRID: AB_2533506), SRY-box transcription factor 2 (SOX2), a marker used for identification of NPC (mouse, 1:500, R&D Systems, Minneapolis, MN, USA, Cat# MAB2018, RRID: AB_358009), Nestin, a marker used for identification of NPC (rabbit, 1:1000, Millipore, Darmstadt, Germany, Cat# ABD69, RRID: AB_2744681), microtubule-associated protein 2 (MAP2), a marker of dendrites (rabbit, 1:1000, Millipore, Burlington, MA, USA, Cat# AB5622, RRID: AB_91939), MAP2 (mouse, 1:1000, Millipore, Cat# AMAb91375, RRID: AB_2716657), beta III tubulin (TUJ1), a neuronal marker (mouse, 1:1000, Sigma-Aldrich, Cat# T8660, RRID: AB_477590), anti-human nuclei (HuNu), a marker of nuclei in human cells (mouse, 1:500, Millipore, Cat# MAB1281, RRID: AB_94090), anti-vesicular glutamate transporter 1 (VGLUT1), a glutamatergic neuronal marker (mouse, 1:250, Millipore, Cat# MAB5502, RRID: AB_262185), gamma-aminobutyric acid (GABA), a GABAergic neuronal marker (rabbit, 1:1000, Sigma-Aldrich, Cat# A2052, RRID: AB_477652), tyrosine hydroxylase (TH), a dopaminergic neuronal marker (rabbit, 1:1000, Millipore, Cat# AB152, RRID: AB_390204), TH (chicken, 1:500, Millipore, Cat# AB9702, RRID: AB_570923), FOXA2, a midbrain neuronal marker (rabbit, 1:500, Cell Signaling Technology, Danvers, MA, USA, Cat# 8186, RRID: AB_10891055), and paired like homeodomain 3 (PITX3), another midbrain neuronal marker (rabbit, 1:500, Millipore, Cat# AB5722, RRID: AB_91997).

Techniques: Marker, Immunostaining, Derivative Assay, Comparison, Modification, Inhibition, Expressing, Infection, Cell Culture, Immunofluorescence, Staining, Two Tailed Test

Journal: Prostate cancer and prostatic diseases

Article Title: SOX2 expression in the developing, adult, as well as diseased prostate

doi: 10.1038/pcan.2014.29

Figure Lengend Snippet: The level of SOX2 was knocked down in PC3 cells and protein lysate was prepared from parental PC3 cells and PC3 cells that have stably integrated with control shRNA, or with variant shSOX2 construct. Beta-Actin was used as loading control.

Article Snippet: The shSOX2 constructs were purchased from Origene (Rockville, MD).

Techniques: Stable Transfection, Control, shRNA, Variant Assay, Construct

Journal: Signal Transduction and Targeted Therapy

Article Title: Focal adhesion kinase/Src family kinase axis-mediated tyrosine phosphorylation of metabolic enzymes facilitates tumor metastasis

doi: 10.1038/s41392-025-02395-5

Figure Lengend Snippet: ACLY and ALDOA tyrosine phosphorylation critically contributes to the activation of c-Myc, Oct4, Sox2, and specific downstream molecules. a PCR was used to evaluate the expression of the AXL , GALK1 , SDC2 , ITGA5 , FGF2 , HIF1A , MMP14 , TWIST1 , VEGFA , and VEGFC genes in metastatic KYSE410 cells harboring ACLY Y542A with or without OAA (1 μM), ALDOA Y174A, Y302A, or Y328A with or without G3P (1 μM) ( n = 3). b c-Myc, Oct4 and Sox2-bound promoter sequences of AXL , GALK1 , SDC2 , or ITGA5 from -85--42, -95--43, -81--31, and -156--110 are indicated. c ChIP was used to assess the ability of c-Myc, Oct4, and Sox2 to bind to the AXL , GALK1 , SDC2 , or ITGA5 promoter regions in metastatic KYSE410 cells harboring ACLY Y542A with or without OAA (1 μM), ALDOA Y174A, Y302A, or Y328A with or without G3P (1 μM) ( n = 3). The data are presented as the means ± SDs

Article Snippet: The transcriptional activity of c-Myc, Sox2, and Oct4 in the lymph nodes was assessed via a human c-Myc activity assay (Raybiotech; Cat # TFEH-CMYC), Sox2 activity assay (Raybiotech; Cat # TFEH-SOX2), and Oct4 activity assay (Raybiotech; Cat # TFEH-Oct4) kits, respectively.

Techniques: Phospho-proteomics, Activation Assay, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Focal adhesion kinase/Src family kinase axis-mediated tyrosine phosphorylation of metabolic enzymes facilitates tumor metastasis

doi: 10.1038/s41392-025-02395-5

Figure Lengend Snippet: Quercetagitrin inhibits the lymph node metastasis of ESCC cells by blocking ALDOA activity. a Protocol used to establish the xenograft model. A lymph node metastasis model was used in the present study. Adobe Illustrator 2022 software was used to construct Fig. 7a. b After the footpads were inoculated with KYSE410 or KYSE510 cells for ~1 week, the mice were intratumorally injected with AAV8-CMV-ALDOA Y174/302/328 A into primary sites and orally treated with quercetagitrin ( n = 5; 25 mg/kg/day, p.o.). After treatment, the lymph nodes were extracted, and the volumes of the lymph nodes were calculated and quantified. Representative images of H&E-stained lymph nodes are shown. Magnification, 1× as indicated. Primary tumors ( c ) or lymph nodes ( d ) from the KYSE410 and KYSE510 tumor groups were extracted, and an IHC assay was subsequently used to evaluate the expression of MCM3, MCM4, MCM5, MCM6, MCM7, or CDC45 in primary tumors ( c ) and the expression of AXL, GALK1, ITGA5, or SDC2 in lymph nodes ( d ). Representative images of IHC staining of the indicated proteins in primary sites or lymph nodes from the KYSE410 (left panel) or KYSE510 (right panel) tumor groups are displayed. Magnification, 20× as indicated. e Primary ESCC tumors were lysed, protein lysates were prepared, and the expression of CDK7, CDK9, Ki67, or LYVE1 was evaluated via quantitative ELISA ( n = 3). f Lymph nodes were lysed, and nuclear proteins were obtained and subjected to the transcriptional activity of c-Myc, Oct4, and Sox2 ( n = 3). The data are presented as the means ± SDs. n.s. not significantly different; * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The transcriptional activity of c-Myc, Sox2, and Oct4 in the lymph nodes was assessed via a human c-Myc activity assay (Raybiotech; Cat # TFEH-CMYC), Sox2 activity assay (Raybiotech; Cat # TFEH-SOX2), and Oct4 activity assay (Raybiotech; Cat # TFEH-Oct4) kits, respectively.

Techniques: Blocking Assay, Activity Assay, Software, Construct, Injection, Staining, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay