Journal: Fluids and barriers of the CNS

Article Title: Neurogenesis and glial impairments in congenital hydrocephalus: insights from a BioGlue-induced fetal lamb model.

doi: 10.1186/s12987-025-00630-3

Figure Lengend Snippet: Fig. 3 Expressions and distributions of Sox2, Vimentin, Aq4, MOBP, MOG and MBP in HCP. A Sox2 gene and protein expressions were higher in HCP compared to control group at E105. However, as gestation progressed, its expression notably decreased. B Upregulation of Vimentin and Aq4 relative expressions were seen, particularly after E125. Western blot analysis showed significantly higher Vimentin protein level in the hydrocephalic groups at E140. C MOBP and MOG relative expressions upregulated at E105 but after E125, their gene expressions and MBP protein level decreased significantly (Data in the bar graphs represent mean ± SEM, green bars: control, blue bars: HCP) (*p < 0.05, **p < 0.01). D Immunostainings of Sox2 (green), Vimentin (red), MBP (magenta) and DAPI in control and hydrocephalic brains at E105, E125 and E140 (× 20, scale bar 50 μm)

Article Snippet: Blots were treated with primary antibodies against Pax6 (Abcam, ab5790, 1:1000), Sox2 (NovusBio, NB110-37235, 1:2000), GFAP (NovusBio, NBP1-05198, 1:5000), Vimentin (Sigma, V6630, 1:200), Olig2 (Millipore, ABE1024, 1:1000), MBP (AvesLab, MBP89927985, 1:2000) and DCX (Invitrogen, PA5-17428, 1:1000) overnight at + 4 °C, followed by a one-hour treatment with secondary antibodies (anti-rabbit IgG HRP-linked, Cell Signaling Technology, 7074S, 1:1000; anti-mouse IgG HRP-linked, Cell Signaling Technology, 7076S, 1:1000; donkey anti-chicken IgY H&L (HRP), Abcam, ab16349, 1:2000 and goat anti-Guinea pig IgG H&L (HRP), Abcam, ab97155, 1:10000) at room temperature.

Techniques: Control, Expressing, Western Blot

Journal: Fluids and barriers of the CNS

Article Title: Neurogenesis and glial impairments in congenital hydrocephalus: insights from a BioGlue-induced fetal lamb model.

doi: 10.1186/s12987-025-00630-3

Figure Lengend Snippet: Fig. 6 Alterations in neurogenesis and gliogenesis in fetal-onset hydrocephalus. During normal early neurodevelopment, neuroepithelial cells (NECs) in the neural tube differentiate into multipotent apical radial glial cells (aRGs) in the VZ. Subsequently, asymmetric divisions of aRGs generate basal progenitors (BPs) in the SVZ, including basal radial glial cells (bRGs) and basal intermediate progenitors (bIPs), which contribute to the production of cortical neurons and macroglia, essential for the development of the neocortex and formation of cortical folds. In this research on hydrocephalus in fetal lambs, disruptions in the VZ were observed as early as E105. Initial compensatory mechanisms occurred through increased proliferation of the NSCs and NPCs, followed by decreases in important neurogenic regulators such as Pax6 and Sox2, particularly at term. Notably, this surge was associated with significant upregulation of DCX, suggesting active neurogenesis at this early stage. However, this proliferation was accompanied by a decline in the expression of critical neurogenic markers such as Pax6 and Sox2, particularly noticeable towards the term, indicating compromised neuronal repair capabilities. Additionally, alterations in the expression of Ascl1 and Tbr2 pointed to disrupted neuronal differentiation pathways, with fluctuating expression patterns evident across various gestational stages. The progression of hydrocephalus was also marked by increased expression of astroglial markers (GFAP, Vimentin, and Aq4), along with pronounced myelin damage indicated by the reduced expressions of MOBP, MOG, and Sox10 as the gestation advanced. Collectively, these findings suggest that surgical intervention between E105 and E125 may offer a critical therapeutic window, providing a potentially effective period for prenatal surgery to mitigate the irreversible damage due to hydrocephalus (created on BioRender.com)

Article Snippet: Blots were treated with primary antibodies against Pax6 (Abcam, ab5790, 1:1000), Sox2 (NovusBio, NB110-37235, 1:2000), GFAP (NovusBio, NBP1-05198, 1:5000), Vimentin (Sigma, V6630, 1:200), Olig2 (Millipore, ABE1024, 1:1000), MBP (AvesLab, MBP89927985, 1:2000) and DCX (Invitrogen, PA5-17428, 1:1000) overnight at + 4 °C, followed by a one-hour treatment with secondary antibodies (anti-rabbit IgG HRP-linked, Cell Signaling Technology, 7074S, 1:1000; anti-mouse IgG HRP-linked, Cell Signaling Technology, 7076S, 1:1000; donkey anti-chicken IgY H&L (HRP), Abcam, ab16349, 1:2000 and goat anti-Guinea pig IgG H&L (HRP), Abcam, ab97155, 1:10000) at room temperature.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Progesterone Receptor Membrane Component 1 suppresses the p53 and Wnt/β-catenin pathways to promote human pluripotent stem cell self-renewal

doi: 10.1038/s41598-018-21322-z

Figure Lengend Snippet: Expression of 108-B6 and 4A68 antigens is localized to undifferentiated and pluripotent hPSCs. ( a ) Multi-color flow cytometric analysis of hPSCs. H9 hPSCs were stained with MAbs (108-B6 or 4A68) and PE-conjugated anti-mouse IgG. The cells were then stained with anti-TRA-1-81 or anti-SSEA3 antibodies followed by incubation with FITC-conjugated anti-mouse IgM or anti-rat IgM, respectively, before analysis. ( b ) Multi-color intracellular flow cytometric analysis of hPSCs. H9 hPSCs were stained with MAbs 108-B6 or 4A68 and PE-conjugated anti-mouse IgG. The cells were fixed with 2% PFA and incubated in PBA containing 0.5% saponin. The cells were then stained with anti-OCT4 or anti-SOX2 antibodies followed by incubation with Alexa 488-cojugated anti-rabbit IgG before analysis.

Article Snippet: Primary antibodies used were mouse MAbs against PGRMC1, NANOG, β-actin, Akt1/2/3, GSK-3β, ERK1/2, CDC2, (all from Santa Cruz Biotechnology), and S6, cyclin E (Cell Signaling Technology), and rabbit polyclonal antibodies against PGRMC1, OCT4, p-Akt1/2/3, β-catenin, cyclin A, cyclin B1, p-CDC2(T14/Y15), CDC25C (all from Santa Cruz Biotechnology), PGRMC1 (GeneTex), SOX2, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Caspase-3 (all from CUSABIO, Wuhan, China), LC3B (Novus Biologicals, Littleton, CO), p62 (Abcam, Cambridge, UK), cyclin D1, p-S6 (S240/244), p-GSK-3β (S9), p-ERK1/2 (T202/Y204), p-β-catenin (S33/37/T41), p-CDC2 (T161), p-CDC25C (S216), p-CDC25C (T48/198), γH2AX (S137), Plk1, p53, p-p53 (S15), p-p53 (S20) (all from Cell Signaling Technology), and anti-acetyl-p53 (K373) (Millipore).

Techniques: Expressing, Staining, Incubation

Journal: Scientific Reports

Article Title: Progesterone Receptor Membrane Component 1 suppresses the p53 and Wnt/β-catenin pathways to promote human pluripotent stem cell self-renewal

doi: 10.1038/s41598-018-21322-z

Figure Lengend Snippet: PGRMC1 knockdown impairs hPSC self-renewal and proliferation. ( a ) Knockdown of PGRMC1 in H9 hPSCs. H9 cells were transfected with either control siRNA (siCon) or PGRMC1 siRNA (siPgrmc1) for 72 h, and subjected to Western blot analysis with 108-B6 and 4A68, followed by HRP-conjugated anti-mouse IgG. Full-length blots are presented in Supplementary Figure . ( b ) Flow cytometric analysis of cell surface PGRMC1 and TRA-1-81 on the surface of H9 cells transfected with siCon or siPgrmc1. Transfected-H9 cells were incubated with 108-B6, 4A68, α-PGRMC1 or anti-TRA-1-81 antibodies, and detected by appropriate FITC-conjugated secondary antibodies. ( c ) Graphic presentation of cell surface expression of PGRMC1 and TRA-1-81 in siCon or siPgrmc1 knockdown hESCs. Relative expression of cell surface PGRMC1 and TRA-1-81 was measured by MFIs of flow cytometry and normalized for control secondary antibody binding. The graph represents the mean values of three independent determinations ±SD (n = 3; * p < 0.05; ** p < 0.01; *** p < 0.005). ( d ) AP staining of siCon or siPgrmc1 knockdown hPSCs. H9 cells were pre-treated with Y-27632, dissociated into single cells, and transfected with siCon or siPgrmc1. After culturing for 7 days, visible colonies were stained with AP assay kit. ( e ) Statistical analysis of AP activity of control or PGRMC1 knockdown hPSCs. AP activity of AP-positive colonies was measured at OD405 (n = 7; *** p < 0.005). ( f ) Western blot analysis showing the expression levels of OCT4, NANOG, SOX2, and PGRMC1 in control or PGRMC1 knockdown hPSCs. β-actin was used as internal protein control and loading control. Full-length blots are presented in Supplementary Figure . ( g ) Cell proliferation of control or PGRMC1 knockdown hESCs. Cell numbers of control or PGRMC1 knockdown hPSCs were determined by trypan blue exclusion assay (n = 7; ** *p < 0.005). ( h ) Cell cycle distribution of control or PGMC1 knockdown hPSCs. Each bar represents mean values of four independent experiments ± SD (n = 4; * p < 0.05; ns, not significant). In ( a , f ), images are representative of at least three independent experiments.

Article Snippet: Primary antibodies used were mouse MAbs against PGRMC1, NANOG, β-actin, Akt1/2/3, GSK-3β, ERK1/2, CDC2, (all from Santa Cruz Biotechnology), and S6, cyclin E (Cell Signaling Technology), and rabbit polyclonal antibodies against PGRMC1, OCT4, p-Akt1/2/3, β-catenin, cyclin A, cyclin B1, p-CDC2(T14/Y15), CDC25C (all from Santa Cruz Biotechnology), PGRMC1 (GeneTex), SOX2, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Caspase-3 (all from CUSABIO, Wuhan, China), LC3B (Novus Biologicals, Littleton, CO), p62 (Abcam, Cambridge, UK), cyclin D1, p-S6 (S240/244), p-GSK-3β (S9), p-ERK1/2 (T202/Y204), p-β-catenin (S33/37/T41), p-CDC2 (T161), p-CDC25C (S216), p-CDC25C (T48/198), γH2AX (S137), Plk1, p53, p-p53 (S15), p-p53 (S20) (all from Cell Signaling Technology), and anti-acetyl-p53 (K373) (Millipore).

Techniques: Knockdown, Transfection, Control, Western Blot, Incubation, Expressing, Flow Cytometry, Binding Assay, Staining, Activity Assay, Trypan Blue Exclusion Assay

Journal: Oncotarget

Article Title: Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cells

doi:

Figure Lengend Snippet: The effects of CD9 silencing on stem cell markers were analyzed in three CD9 -silenced GSC lines (“CD9-si”), as compared to the non- CD9 -silenced cells (“control”). A. Gene expression of stem cell markers nestin, SOX2 and CD133 was measured by qPCR in three GSC lines NCH644, NCH421k and NCH660h. B. Nestin, SOX2 and CD133/1 protein expression analysis in the CD9 -silenced NCH644 cell line. Blue, cell nuclei; green, proteins, as indicated. Representative images of three repeated experiments are shown for both channels (blue, green) as well as for overlays. Scale bar, 100 μm. C. Immunocytochemistry detection in the CD9 silenced GSC lines NCH644 and NCH421k was performed for detection of differentiation protein markers, such as glial fibrillary acidic protein (GFAP) and b-III-tubulin, related to glial and neuronal differentiation, respectively as well as for detection of stem cell markers, such as CD133/1 and SOX2. Representative images of two repeated experiments are shown. Scale bar, 100 μm. Quantification of the results is shown on the right panel. Two visible fields of each staining were evaluated at 200 × magnification. Data are means ±SD of two independent experiments. D. Clonogenic assay for self-renewal of the CD9 -silenced GSC lines. Data are means ± SD of three independent experiments. Representative data of each cell line are also shown. Scale bar, 100 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The following anti-human primary antibodies were applied for 1 h at room temperature: anti-nestin (clone 10C2, 1:200; Millipore), anti-SOX2 (clone EPR3131, 1:50; Abgent), anti-Ki-67 (clone EPR3610, 1:200; Abcam), and anti-CD9 (clone MEM-61, 1:50; Abcam).

Techniques: Expressing, Immunocytochemistry, Staining, Clonogenic Assay

Journal: Oncotarget

Article Title: Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cells

doi:

Figure Lengend Snippet: Spheroids from CD9 -silenced NCH644 and NCH421k (“CD9-si”) and the non- CD9 -silenced cells (“control”) were implanted into the brains of nude rats to determine the effects of CD9 silencing on survival. A. Kaplan–Meier curves for CD9 -silenced GSC lines compared to the non- CD9 -silenced GSC lines. B. Representative immunohistochemistry staining for expression of CD9, proliferation marker Ki-67, stem cell markers nestin and SOX2. Scale bar, 200 μm. C. Quantification of immunohistochemistry staining of paraffin sections from CD9 -silenced and non- CD9 -silened xenografts. Complete expression scores were calculated as mean percentage of positive cells throughout the whole tumor area multiplied by the mean staining intensity (0, negative; 1, weak; 2, moderate; 3, strong). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The following anti-human primary antibodies were applied for 1 h at room temperature: anti-nestin (clone 10C2, 1:200; Millipore), anti-SOX2 (clone EPR3131, 1:50; Abgent), anti-Ki-67 (clone EPR3610, 1:200; Abcam), and anti-CD9 (clone MEM-61, 1:50; Abcam).

Techniques: Immunohistochemistry, Staining, Expressing, Marker

Journal: Oncology Letters

Article Title: Macrophages activate mesenchymal stem cells to acquire cancer-associated fibroblast-like features resulting in gastric epithelial cell lesions and malignant transformation in vitro

doi: 10.3892/ol.2018.9703

Figure Lengend Snippet: Gastric epithelial cells incubated with CM from macrophage-hucMSCs exhibit several properties of stem cells. (A) GES-1 cells were incubated with CM from hucMSCs or macrophage-hucMSCs for 48 h, and then seeded in non-adherent culture conditions for spheroid formation. Depicted are representative images of spheroid colonies after 15 days. Magnification, ×400; scale bar, 50 µm. (B) Quantification of the soft agar colonies shown in (A). (C) Western blot analysis of Nanog, SOX2, and BMI-1 in GES-1 cells (the controls) and GES-1 cells co-cultured with CM from hucMSCs and macrophage-hucMSCs after 48 h. The expression of all analyzed proteins is significantly enhanced in the GES-1 cells cultured with CM from macrophage-hucMSCs, compared with the control. (D) Representative image of single-colony formations of GES-1 cells incubated with CM from hucMSCs or CM from macrophage-hucMSCs. (E) The number of colonies is depicted as mean ± standard deviation. *P<0.05, # P<0.01; GES-1 cells treated with medium only served as the control (magnification, ×100). Macrophage-hucMSC, human umbilical cord-derived mesenchymal stem cells pre-cultured with macrophages for 48 h; CM, conditioned medium; SOX2, SRY-box 2; BMI-1, polycomb complex protein BMI-1.

Article Snippet: The membranes were incubated with 5% skimmed milk to block non-specific protein at room temperature for 1 h. Membranes were incubated overnight at 4°C with primary antibodies at a dilution of 1:800 for rabbit polyclonal anti-N-cadherin (cat. no. BS2224; Bioworld Technology, Inc., St. Louis Park, MN, USA), 1:1,000 for rabbit polyclonal anti-E-cadherin (cat. no. BS1098; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-vimentin (cat. no. BS1855; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-α-SMA (cat. no. BS8796; Bioworld Technology, Inc.), 1:800 for rabbit polyclonal anti-B-cell lymphoma-2 (Bcl-2) (cat. no. BS70205; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-Bcl-2-associated X (Bax) (cat. no. BS1030; Bioworld Technology, Inc.), 1:500 for rabbit polyclonal anti-SOX2 (cat. no. BS6161; Bioworld Technology, Inc.), 1:1,000 for rabbit polyclonal anti-Bmi-1 (cat. no. BS6015; Bioworld Technology, Inc.), 1:10,000 for rabbit polyclonal anti-β-actin (cat. no. AP0060; Bioworld Technology, Inc.), 1:1,000 for mouse monoclonal anti-FAP (cat. no. sc-71094; Santa Cruz Biotechnology, Inc.,) and 1:1,000 for rabbit polyclonal anti-Nanog (cat. no. sc-33759; Santa Cruz Biotechnology, Inc.).

Techniques: Incubation, Western Blot, Cell Culture, Expressing, Standard Deviation, Derivative Assay