Journal: International Journal of Molecular Sciences

Article Title: EN1 Regulates Cell Growth and Proliferation in Human Glioma Cells via Hedgehog Signaling

doi: 10.3390/ijms23031123

Figure Lengend Snippet: Elevated EN expression is associated with glioma cancer progression. ( A ) RNA-seq results for mouse glioma stem cells lines vs. neural stem cells (left) and for mouse brain tumor tissues vs. normal brain tissues (right). ( B ) The expression of EN1 (left) and EN2 (right) in glioma samples from TCGA database. Black, blue and red color is for Grade 2, 3 and 4 respectively. ( C ) Analysis using the Henry Ford Hospital dataset, showing correlation between the EN1 (left) and EN2 (right) gene expression levels and glioma patient tumor grade (○ for Non-tumor, □ and ▲ for Oligodendroglioma-2 and 3, ◊ and ∆ for Asreocytoma-2 and 3, red ● for GBM). ( D ) Correlation between glioma patient survival and the EN1 expression level from TCGA glioblastoma database (left) and the Chinese Glioma genome Atlas gliomas database (right). ( E ) For the indicated glioma cell lines, Engrailed 1 expression assessed by qPCR (left) and Engrailed 1 accumulation assessed by immunoblotting (right). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns (no significant) according to Student’s t -test.

Article Snippet: The membrane was incubated (overnight, 4 °C) with the following primary antibodies: (EN1-2377, NIBS, Beijing, China), (EN1-2378, NIBS, Beijing, China), (EN1 Polyclonal Antibody, Invitrogen, Waltham, MA, USA), (EN1-4D9, University of California, USA), (EN1-4G11, Columbia University, USA), (EN1, Abcam, Cambridge, MA, USA), (EN1, Sigma-Aldrich, USA), (EN1, Atlas Antibodies, Stockholm, Sweden), (EN1, Cusabio, Wuhan, China) (only EN1-2377 recognizes the correct size of EN1 band), (GAPDH, Origene, USA), and (TULP3, Abcam, Cambridge, MA, USA), (Gli1, Cell Signaling Technology, Danvers, MA, USA), before washing three times with TBST.

Techniques: Expressing, RNA Sequencing Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: EN1 Regulates Cell Growth and Proliferation in Human Glioma Cells via Hedgehog Signaling

doi: 10.3390/ijms23031123

Figure Lengend Snippet: EN1 knockdown restrains the proliferation and migration of glioma cells. ( A ) Assessment of knockdown efficiency for EN1 with immunoblotting, and confirmation of EN1 knockout in U251 cells with immunoblotting and genome sequencing. Cell proliferation was assessed using CellTiter-Glo assays ( B ), 5-ethynyl-2′-deoxyuridine (Edu) assays ( C ), and colony formation assays ( D ) (● for shcon, ■ for shEN1-3898, ▲ for shEN1-3899, ◆ for shEN1-3901). ( E ) Cell migration was assessed with a wound-healing assay. ( F ) Quantification of wound-healing assay. ( G ) Survival curves for mice bearing xenograft tumors derived from shRNA-EN1 cells or scramble control cells. ( H ) Cell proliferation was assessed by CellTiter-Glo and colony formation assays in U-87 MG cells after EN1 overexpression. Scale bar: 100 μm. Data are presented as the mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 according to Student’s t -test.

Article Snippet: The membrane was incubated (overnight, 4 °C) with the following primary antibodies: (EN1-2377, NIBS, Beijing, China), (EN1-2378, NIBS, Beijing, China), (EN1 Polyclonal Antibody, Invitrogen, Waltham, MA, USA), (EN1-4D9, University of California, USA), (EN1-4G11, Columbia University, USA), (EN1, Abcam, Cambridge, MA, USA), (EN1, Sigma-Aldrich, USA), (EN1, Atlas Antibodies, Stockholm, Sweden), (EN1, Cusabio, Wuhan, China) (only EN1-2377 recognizes the correct size of EN1 band), (GAPDH, Origene, USA), and (TULP3, Abcam, Cambridge, MA, USA), (Gli1, Cell Signaling Technology, Danvers, MA, USA), before washing three times with TBST.

Techniques: Migration, Western Blot, Knock-Out, Sequencing, Wound Healing Assay, Derivative Assay, shRNA, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: EN1 Regulates Cell Growth and Proliferation in Human Glioma Cells via Hedgehog Signaling

doi: 10.3390/ijms23031123

Figure Lengend Snippet: EN1 knockdown increased intracellular ROS levels and elevated susceptibility to γ-ray irradiation. ( A ) EN1 knockdown elevated the intracellular ROS levels of glioma cells, assessed with H2DCFDA assays (● for shcon, ■ for shEN1-3898, ▲ for shEN1-3899, ◆ for shEN1-3901). ( B ) Colony forming assays demonstrating that EN1 knockdown elevated the susceptibility of glioma cells to γ-ray irradiation, assessed with plating efficiency (PE), where PE is the fraction of colonies counted divided by cells plated without radiation. Data are presented as the mean from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 according to Student’s t -test.

Article Snippet: The membrane was incubated (overnight, 4 °C) with the following primary antibodies: (EN1-2377, NIBS, Beijing, China), (EN1-2378, NIBS, Beijing, China), (EN1 Polyclonal Antibody, Invitrogen, Waltham, MA, USA), (EN1-4D9, University of California, USA), (EN1-4G11, Columbia University, USA), (EN1, Abcam, Cambridge, MA, USA), (EN1, Sigma-Aldrich, USA), (EN1, Atlas Antibodies, Stockholm, Sweden), (EN1, Cusabio, Wuhan, China) (only EN1-2377 recognizes the correct size of EN1 band), (GAPDH, Origene, USA), and (TULP3, Abcam, Cambridge, MA, USA), (Gli1, Cell Signaling Technology, Danvers, MA, USA), before washing three times with TBST.

Techniques: Irradiation

Journal: International Journal of Molecular Sciences

Article Title: EN1 Regulates Cell Growth and Proliferation in Human Glioma Cells via Hedgehog Signaling

doi: 10.3390/ijms23031123

Figure Lengend Snippet: EN1 increases the activity of proliferation related Hedgehog signaling pathways. ( A ) Overrepresented Gene Ontology (GO) terms from RNA−seq analysis of upregulated gene sets and downregulated gene sets in EN1 knockdown group compared to control cells. ( B ) Immunoblotting assays showing that EN1 knockdown significantly reduces the Gli1 level in glioma cells. ( C ) The EN1 and Gli1 transcript levels in U251 cells transduced with scramble control, shEN1, shcon + Gli1, or shEN1 + Gli1. ( D ) Overexpression of Gli1 rescues the restrained colony-forming abilities caused by the knockout of EN1 (□ for U251-sgLacZ, ● for U251-sgEN1, ■ for U251+Gli1-sgEN1). Data are presented as the mean from three independent experiments. * p < 0.05, ### p < 0.001 according to Student’s t -test.

Article Snippet: The membrane was incubated (overnight, 4 °C) with the following primary antibodies: (EN1-2377, NIBS, Beijing, China), (EN1-2378, NIBS, Beijing, China), (EN1 Polyclonal Antibody, Invitrogen, Waltham, MA, USA), (EN1-4D9, University of California, USA), (EN1-4G11, Columbia University, USA), (EN1, Abcam, Cambridge, MA, USA), (EN1, Sigma-Aldrich, USA), (EN1, Atlas Antibodies, Stockholm, Sweden), (EN1, Cusabio, Wuhan, China) (only EN1-2377 recognizes the correct size of EN1 band), (GAPDH, Origene, USA), and (TULP3, Abcam, Cambridge, MA, USA), (Gli1, Cell Signaling Technology, Danvers, MA, USA), before washing three times with TBST.

Techniques: Activity Assay, RNA Sequencing Assay, Western Blot, Transduction, Over Expression, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: EN1 Regulates Cell Growth and Proliferation in Human Glioma Cells via Hedgehog Signaling

doi: 10.3390/ijms23031123

Figure Lengend Snippet: Silencing EN1 upregulates the expression of TULP3. ( A ) Volcano plot of differentially expressed genes (DEGs) in EN1 knockdown glioma cells (vs. scramble control cells). qPCR ( B ) and immunoblotting ( C ) confirmed that EN1 knockdown glioma cells have elevated TULP3. ( D ) Staining of glioma cells with antibodies against ARL13B and acetylated tubulin. ( E ) EN1 knockdown significantly decreased the number of primary cilia. Scale bar: 50 µm. Data are presented as the mean from three independent experiments (● for shcon, ■ for shEN1-3898, ▲ for shEN1-3899, ◆ for shEN1-3901). * p < 0.05 according to Student’s t -test.

Article Snippet: The membrane was incubated (overnight, 4 °C) with the following primary antibodies: (EN1-2377, NIBS, Beijing, China), (EN1-2378, NIBS, Beijing, China), (EN1 Polyclonal Antibody, Invitrogen, Waltham, MA, USA), (EN1-4D9, University of California, USA), (EN1-4G11, Columbia University, USA), (EN1, Abcam, Cambridge, MA, USA), (EN1, Sigma-Aldrich, USA), (EN1, Atlas Antibodies, Stockholm, Sweden), (EN1, Cusabio, Wuhan, China) (only EN1-2377 recognizes the correct size of EN1 band), (GAPDH, Origene, USA), and (TULP3, Abcam, Cambridge, MA, USA), (Gli1, Cell Signaling Technology, Danvers, MA, USA), before washing three times with TBST.

Techniques: Expressing, Western Blot, Staining

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , Schematic diagram of the 4 DIV CNP differentiation protocol. b , Representative immunostaining images of H9 and GBX2 -/- CNPs at 4 DIV showing OTX2 - /CDX2 + cells. A few 4X CNPs were positive for OTX2, but no cells were positive for CDX2. Scale bars, 20 µm. c-d , qPCR analysis of OTX2 (c) and CDX2 (d) expression in H9, GBX2 -/- and 4X CNPs at 4 DIV. The data are presented as the mean ± SD; n= 3. One-way ANOVA showed statistical significance, and then an unpaired t-test comparing two groups was performed. *P< 0.05; **P< 0.01. e , Heatmap of the expression of pluripotent and neural genes representing the anterior, midbrain, hindbrain and spinal cord regions in H9, GBX2 -/- and 4X CNPs at 4 DIV. f , The top 10 downregulated (blue) and upregulated (red) genes (and additional selected genes in bold) between cultured 4X and H9 cells, cultured GBX2 -/- and H9 cells and cultured 4X and GBX2 -/- cells at 4 DIV. The threshold bar (white line) indicates a fold change of ±2. g , Schematic diagram of the 11 DIV CNP differentiation protocol. h , RNA expression analysis of the midbrain genes (orange) OTX2, EN1 and PAX8 ; the hindbrain genes (gray) MAFB, EGR2, HOXA2, HOXB1, HOXA3, HOXB2 , and HOXA4 ; and the spinal cord genes (purple) HOXB8 and HOXC10 . The data are presented as the mean ± SD; n= 3. One-way ANOVA followed by Tukey’s multiple comparisons test. *P< 0.05; **P< 0.01. i , Representative immunohistochemical analysis of OTX2/EN1 double-positive cells among 11 DIV 4X cells. No OTX2/EN1 double-positive cells were detected among H9 cells. Scale bars, 10 µm. For (b) and (i), DAPI was used as a nuclear stain. Caudal neural progenitor: CNP.

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: Immunostaining, Expressing, Cell Culture, RNA Expression, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , Schematic diagram of the 11 DIV CNP differentiation protocol using different concentrations of GSK3i ranging from 0.7 µM to 3 µM. b , Flow cytometry analysis of the percentage of OTX2-positive cells among H9, GBX2 -/- and 4X CNPs at 11 DIV. The data are presented as the mean ± SD; n= 3. Two-way ANOVA followed by Tukey’s multiple comparisons test comparing groups treated with the same concentration of GSK3i. **P< 0.01. c , Schematic diagram of the 16 DIV caudal midbrain differentiation protocol using different concentrations of GSK3i ranging from 0.5 µM to 1 µM. d , Quantification of OTX2-positive cells among H9 and 4X cells at 16 DIV after administration of GSK3i at concentrations ranging from 0.5 µM to 1 µM. The data are presented as the mean ± SD; n= 4-8. Two-way ANOVA followed by Sidak’s multiple comparisons test comparing groups treated with the same concentration of GSK3i. **P< 0.01. e , Expression of OTX2, EN1, LMX1A, CNPY1 , and HOXA2 in H9 and 4X cells treated with a range of GSK3i concentrations at 16 DIV. The data are presented as the mean ± SD., n= 3. Two-way ANOVA followed by Sidak’s multiple comparisons test comparing groups treated with the same concentration of GSK3i. *P< 0.05; **P< 0.01. f , Representative immunohistochemical analysis of OTX2, EN1, FOXA2, and LMX1A expression in H9 and 4X cells treated with GSK3i at concentrations of 0.6 µM and 1 µM on 16 DIV. DAPI was used as a nuclear stain. Scale bars, 50 µm. Caudal neural progenitor: CNP.

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: Flow Cytometry, Concentration Assay, Expressing, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , UMAP of cultured 4X and H9 cells at 16 DIV and a graph of the percentage of cells that each cell line contributed to each cluster (n = 10 4X cell spheres; n = 10 H9 cell spheres; total of 4,682 cells). b , Heatmap of the expression of selected genes illustrating the identity of the clusters. c, Percentage of 4X and H9 cells expressing OTX2 and EN1 . d , Feature plot of the contribution of each cell line to each cluster and feature plot of gene expression levels of OTX2, EN1, LMX1A, FOXA2, FGF8, HOXA/B family members and STMN2 . e , UMAP of cultured 4X and H9 cells at 62 DIV and a graph of the percentage of cells that each cell line contributed to each cluster (n = 4 4X cell cultures; n = 4 H9 cell cultures; total of 6,804 cells). f , Heatmap of the expression of selected genes illustrating the identity of the clusters. g , Feature plot of the contribution of each cell line to each cluster and feature plot of the gene expression levels of STMN2, DCX, TH, LMX1A, EN1, SHH and COL3A1 .

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: Cell Culture, Expressing

Journal: bioRxiv

Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

doi: 10.1101/2021.09.28.462222

Figure Lengend Snippet: a , Overview of the in vivo study. Unilateral 6-OHDA-induced MFB lesions were generated (week -4) and confirmed 3 weeks later by the cylinder and amphetamine-induced rotation tests. The animals were subdivided into 3 groups with similar average scores on the rotation test. Four weeks after lesioning (week 0), two of these subgroups were transplanted with 250,000 cells (H9 or 4X cells), and the third group did not undergo transplantation (6-OHDA lesion group). The rotation test was repeated at weeks 8 and 18 posttransplant, and the cylinder test was repeated at week 18. The animals were killed at week 19 posttransplantation (23 week after lesioning) for histological analysis. b , Amphetamine-induced rotational asymmetry. Two-way repeated measures ANOVA followed by Sidak’s multiple comparison test; time: F(1.689, 35.46) =19.50, P<0.0001; treatment: F (2, 21) = 15.23 P< 0.0001. ** P< 0.01 and ****P< 0.0001 vs. the 4X cell-transplanted group at the same time point. §§ P< 0.01 and §§§§P<0.0001 vs. the same group at week -1. c , The use of each forelimb (contra or ipsi) and both forelimbs in the cylinder test was analyzed by two-way repeated measures ANOVA followed by Sidak’s multiple comparison test with time and group as variables. Time x group: both: F (2, 22) = 5.785, P=0.009; ipsi: F (2, 22) = 8.800, P=0.001; contra: F (2, 22) = 4.642, P=0.021. *P<0.05 and **P<0.01 vs. the same group at -1 week. $P<0.05 and $$P<0.01 vs. the 6-OHDA lesion group at the same time point. £P<0.05 and ££P<0.01 vs. the H9 cell-transplanted group at the same time point. The data in (b) and (c) are presented as the mean ± SEM. n= 7 rats in the 6-OHDA lesion group, n = 9 rats in the 4X cell-transplanted group, and n=8 rats in the H9 cell-transplanted group). d , Representative photos of coronal sections from all three groups immunostained for TH. Higher magnification images of the areas in the frame are shown on the right. Scale bars, 50 µm for all three photos in the column. The graphs on the right show (e) the estimated numbers of TH-positive cells in the grafts, (f) the yield of TH-positive neurons per 100,000 grafted cells and (g) the volume of the TH-positive graft (see Methods for details). h-i , Representative photomicrographs showing HNA-positive and TH-positive cells within H9 cell (h) and 4X cell (i) grafts. The squares in h-i and h’-i’ indicate the magnified areas shown in h’-i’ and h’’-i’’ , respectively. DAPI was used as a nuclear stain. Scale bars, 200 μm (h-i) and 50 μm ( h’-i’) . j-n , Representative immunofluorescence images of cells double-positive for TH/FOXA2 (j) , TH/LMX1A (k) , TH/EN1 (l) , TH/GIRK2 (m) and TH/CALB1 (n) within 4X cell grafts. (j’-n’) High-power images of j-n highlighting the graft composition. Scale bar, 50 μm. o , Quantitative analysis of the immunofluorescence data in m and n, showing the percentages of GIRK2/TH and CALB1/TH double-positive cells within 4X cell grafts. The data are presented as the mean percentage ± SD (n=9 rats).

Article Snippet: The following primary antibodies were applied overnight at 4 °C: goat anti-OTX2 (1:500, R&D Systems, cat# AF1979), mouse anti-CDX2 (1:200, BioGenex, cat# MU392-UC), mouse anti-Engrailed1 (EN1, 1:40, DSHB, cat# 4G11-s), rabbit anti-EN1 (1:50, Merck, cat# HPA073141), rabbit anti-FOXA2 (1:500, Cell Signaling, cat# 8186), goat anti-FOXA2 (1:200, R&D Systems, cat# AF2400), rabbit anti-LMX1A (1:5000, Millipore, cat# AB10533), mouse anti-TH (1:2000, Millipore, cat# MAB318), rabbit anti-TH (1:1000, Pel Freez, cat# P40101-150), rabbit anti-GIRK2 (1:500, Alomone, cat# APC-006), mouse anti-CALB1 (1:5000, SWANT, cat #300), rabbit anti-Collagen3A1 (1:1000, NovusBio, cat# NB120-6580), sheep anti-hCOL1A1 (1:200, R&D Systems, cat# AF6220), and mouse anti-HNA (1:200, Abcam, cat# ab191181).

Techniques: In Vivo, Generated, Transplantation Assay, Staining, Immunofluorescence