Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 maintains BTIC self-renewal. ( a ) Morphology of self-renewing spheres and immunofluorescence of neuronal and glial lineage markers in differentiated cells. Scale bar=200 μm. ( b ) Immunofluorescence of ING5 in undifferentiated (upper panels) and cells differentiated for 5 days (lower panels). Scale bar=20 μm. ( c , d ) The mRNA levels ( c ) and protein levels ( d ) of ING5 decrease during differentiation in BT 189 cells. n =4. ( e ) Immunoblotting of ING5 protein in the BT 12, and BT 134 lines during differentiation. ( f ) ING5 overexpression increases sphere formation rates and average volumes in BTIC sphere formation assays ( n =3, ** P <0.01, * P <0.05). Scale bar=400 μm. ( g ) ING5 knockdown by siRNA decreases sphere formation rates and sphere sizes of BTICs ( n =4, ** P <0.01, * P <0.05). Scale bars=400 μm. ( h ) (Left) Sphere formation rates for cell lines stably expressing shRNAs against ING5 (shR1 and shR2-ING5) or control non-targeting shRNA (shR-ctr). ( n =4, ** P <0.01, * P <0.05). (Right) Fluorescence of the RFP reporter in stable cell lines superimposed with differential interference contrast (DIC) images. Scale bar=100 μm. ( i ) RT-qPCR analysis of stem cell core transcription factors and stem cell markers after ING5 overexpression. ( j , k ) Western blot analysis of the neural stem cell marker Nestin and neuronal lineage marker Tubb3 in response to ING5 overexpression ( j ) and knockdown ( k ).

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Western Blot, Over Expression, Knockdown, Stable Transfection, Expressing, Control, shRNA, Fluorescence, Quantitative RT-PCR, Marker

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 increases the stem cell pool and inhibits differentiation. ( a ) Flow cytometry analysis of CD133/CD44 positive cells in iPB cell lines (top panels) and CD44 positive cells in shRNA cell lines (bottom panels), gated by isotype control. ( b ) (Left) Mitotic pair analysis of the three division modes: symmetric proliferating (sym-pro), symmetric differentiating (sym-diff) and asymmetric (asym) cell division, in iPB cell lines. Over 150 pairs were counted for each group in one experiment. n =3, * P <0.05. (Right) An example of cell division symmetry based on the distribution of stem cell factor Nestin with the Red arrow indicating symmetric differentiating division and the white arrow asymmetric division. ( c ) Morphological changes of iPB cells before (Day 0) and after (Days 1–3) differentiation induced by 1% FBS. Scale bar=100 μm. ( d ) After differentiation for 5 days, immunofluorescence of Nestin and Tubb3 in shRNA cell lines with an RFP reporter. Scale bar=200 μm. ( e ) Western blot of Nestin and Tubb3 in differentiated shRNA cell lines corresponding to cells shown in d .

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Flow Cytometry, shRNA, Control, Immunofluorescence, Western Blot

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: ING5 activates mitogenic pathways to promote self-renewal. ( a ) The sphere formation rates of iPB-ctr and iPB-ING5 overexpressing cells at three successive passages in the absence of EGF and FGF treatment. ( n =3, ** P <0.01, *** P <0.001). ( b ) DIC images of spheres from the tertiary sphere passage in iPB cells. Scale bar=400 μm. ( c ) Sphere formation rate under treatment with protein kinase inhibitors ( n =3, * P <0.05). ( d ) Protein and phosphorylated protein levels of effectors in the PI3K and MEK pathways. Cells were treated with PX-866 at 1 μ M and PD184352 at 2 μ M for 48 h. ( e ) Immunofluorescence of Nestin and Tubb3 in differentiated iPB control (Left panels) or iPB-ING5 cells (Right panels) treated with 1 μ M PX-866 or 2 μ M PD184352. Scale bar=200 μm. ( f ) Flow cytometry analysis of the CD133 positive population in PX-866 (1 μ M ) and PD184352 (2 μ M ) treated BTIC 189 cells, gated by isotype control.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Immunofluorescence, Control, Flow Cytometry

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: The FSH pathway transduces effects of ING5 on stem cell properties. ( a ) Sphere formation assay for cells treated with calcium modulators and FSHR blocking antibody (Anti-FSHR) at the indicated concentrations ( n =3, * P <0.05 and ** P <0.01 compared to iPB-ctr/DMSO; # P <0.05 and ## P <0.01 compared to iPB-ING5/DMSO). ( b ) IPA downstream function analysis indicates the FSH pathway is elevated by ING5. Genes positively correlated with this function were listed with fold changes. ( c ) RT-qPCR of genes related to hormone and steroidogenesis functions. ( n =3, * P <0.05, ** P <0.01) ( d ) The expression levels of FSHB and FSHR genes in BT 189 cells before and after differentiation for 1–5 days. ( e ) Immunostaining for ING5 and FSHR in iPB cells. Scale bar=100 μm. ( f ) Flow cytometry analysis of CD133 positive cells in BT 189 cells treated with FSHR neutralizing antibody or IgG control, gated by isotype control. ( g ) Immunofluorescence of Nestin and Tubb3 shows inhibition of the FSH pathway induces neuronal differentiation. Scale bar=200 μm. ( h ) FSH recombinant protein treatment at indicated concentrations increases sphere formation rates in shRNA cell lines ( n =3, * P <0.05 and ** P <0.01 compared to untreated shR-ctr; # P <0.05 compared to untreated shR-ING cells). ( i ) FSH recombinant protein treatment induces sphere-forming abilities in iPB-ctr cells but not in ING5 overexpressing cells ( n =3, ** P <0.01). ( j ) Sphere formation rates for cells treated with Anti-FSHR or BAPTA alone, and the combination of both ( n =3, * P <0.05). ( k ) FSH treatment at 5 ng/ml for 3 days induces the expression of OCT4 and Nestin in BT 189 cells.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Tube Formation Assay, Blocking Assay, Quantitative RT-PCR, Expressing, Immunostaining, Flow Cytometry, Control, Immunofluorescence, Inhibition, Recombinant, shRNA

Journal: Oncogene

Article Title: ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways

doi: 10.1038/onc.2017.324

Figure Lengend Snippet: PHD motif is required for the function of ING5 in BTICs and ING5 levels negatively correlate with survival of GBM. ( a ) Sphere formation assays in iPB cell lines overexpressing wild-type (ING5-FLAG) and PHD-deleted ING5 (ΔPHD) ( n =3, ** P <0.01). ( b ) Western blot shows the protein levels of endogeneous ING5, wildtype ING5 with a FLAG tag and PHD-deleted ING5 (black arrows) in three iPB cell lines. ( c ) ChIP analysis of ING5 binding to promoters of target genes presented as fold enrichment relative to IgG controls. The endogenous ING5 in BT 189 cells, overexpressed ING5 with a Flag tag in iPB-ING5 cells and overexpressed PHD-deleted ING5 protein with a Flag tag were immunoprecipitated by the ING5 antibody and Flag antibody respectively. The upper panels are the schematic representation of the location of the primer sets and promoter regions enriched for ING5 binding were shown in red. ( d ) Kaplan–Meier survival analysis of TCGA GBM patients with high and low levels of ING5 expression (stratified by mean value, n =114). ( e , f ) ING5 expression levels negatively correlate with survival of the Proneural subtype ( n =24) and the Classical subtype ( n =30) of GBM patients. ( g ) The relationship of ING5 levels to survival in the SOX2-low group of patients (ING5, SOX2 stratified by median values, n =61). ( h ) Model for how ING5 functions in the maintenance of BTIC self-renewal. In the absence of growth factors, ING5 induces FSH and calcium signaling by promoting transcription of the FSH receptor and ligand genes, and various plasma membrane calcium channel genes. The FSH and calcium signaling pathways further activate PI3K/AKT and MEK/ERK signaling to induce stem cell features and the expression of stemness factors OCT4, OLIG2 and Nestin. Gene activation by ING5 is dependent on its PHD motif to target ING5-associated histone acetyltransferase complexes to the promoters.

Article Snippet: For immunofluorescence assays, cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and then incubated with the primary antibodies against Nestin (1: 800; MAB-1259, R&D Systems), Tubb3 (1:400; MRB-435P, Biolegend, San Diego, CA, USA), GFAP (1:200; Z0334, Dako, Glostrup, Denmark) or FSHR (1:30, MAB65591, R&D Systems) at 4 °C overnight as described.

Techniques: Western Blot, FLAG-tag, Binding Assay, Immunoprecipitation, Expressing, Clinical Proteomics, Membrane, Protein-Protein interactions, Activation Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Expression of a SOX1 overlapping transcript in neural differentiation and cancer models

doi: 10.1007/s00018-017-2580-3

Figure Lengend Snippet: Differentiation of the ReN human neuroprogenitor cell line. a Imaging of day 0 (control) and day 6 (differentiated) ReN cultures in brightfield ( left panels ) and following immunostaining for NESTIN and MAP2 ( green ) with corresponding dapi counterstain ( blue ). Bar 50 µm. b Relative quantification (RQ) of SOX1 mRNA expression analysed by quantitative RT-PCR across the different time points of ReN cells differentiation (day 0, 2, 4 and 6). Error bars represent ±RQ, n = 3, *** p < 0.001; **** p < 0.0001

Article Snippet: After PBS wash, samples were incubated with a dilution of primary antibody against NESTIN (1:100, cat. # IC1259F, R&D Systems, Abingdon, UK) or MAP2 (1:100, MT-01, ExBio, Vestec, Czech Republic) overnight at 4 °C, before extensive washing, incubation in a FITC-labelled secondary antibody solution (1:500, FI-2000, Vector laboratories, Peterborough, UK) for 1 h at room temperature, further washing and finally mounting with DAPI-containing Vectashield (H-1200, Vector laboratories).

Techniques: Imaging, Control, Immunostaining, Quantitative Proteomics, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Efficient Derivation of Multipotent Neural Stem/Progenitor Cells from Non-Human Primate Embryonic Stem Cells

doi: 10.1371/journal.pone.0049469

Figure Lengend Snippet: ( A ) Marmoset ESC-derived primary and secondary neurospheres were dissociated and allowed to differentiate for 10 days, followed by immunocytochemical analysis of βIII-tubulin (neurons), GFAP (astrocytes), CNPase (oligodendrocytes) and Nestin (undifferentiated neural cells). Scale bars, 50 µm. ( B ) The proportions of cells positive for each cell type-specific marker are presented as the percentage of total cells counted by Hoechst 33258-stained nuclei. Data are presented as the means ± SEM ( n = 3).

Article Snippet: Then, samples were incubated at 4°C overnight with the following antibodies: anti-βIII tubulin (mouse IgG 2b , 1∶500; Sigma), Alexa488-conjugated anti-βIII tubulin (mouse IgG, 1∶2,000, Covance, Princeton NJ), anti-glial fibrillary acidic protein (GFAP) (rabbit IgG, 1∶10,000, Dako, Glostrup, Denmark), anti-2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) (mouse IgG 1 , 1∶4,000, Sigma), anti-human Nestin (rabbit IgG, 1∶4,000) , anti-myelin basic protein (MBP) (rat IgG, 1∶500, AbD Serotec, Raleigh, NC, USA), anti-O4 (mouse IgM 1∶1,000, Millipore), and anti-platelet-derived growth factor receptor (PDGFR)α (rabbit IgG, 1∶2,000, Santa Cruz, Santa Cruz, CA, USA).

Techniques: Derivative Assay, Marker, Staining

Journal: PLoS ONE

Article Title: Efficient Derivation of Multipotent Neural Stem/Progenitor Cells from Non-Human Primate Embryonic Stem Cells

doi: 10.1371/journal.pone.0049469

Figure Lengend Snippet: NS/PCs derived from EBs treated with both RA and purmorphamine, were transplanted into the right striatum of 6–8-week-old female NOD/SCID mice. 4 weeks after the injection, mice were sacrificed and processed for immunohistochemical analysis with the antibodies against markers for neurons (Hu), astrocytes (GFAP), oligodendrocytes (APC), and neural progenitors (Nestin). The marmoset ESC-derived NS/PCs could give rise to neurons, astrocytes and oligodendrocytes in vivo , as well. Scale bars, 20 µm.

Article Snippet: Then, samples were incubated at 4°C overnight with the following antibodies: anti-βIII tubulin (mouse IgG 2b , 1∶500; Sigma), Alexa488-conjugated anti-βIII tubulin (mouse IgG, 1∶2,000, Covance, Princeton NJ), anti-glial fibrillary acidic protein (GFAP) (rabbit IgG, 1∶10,000, Dako, Glostrup, Denmark), anti-2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) (mouse IgG 1 , 1∶4,000, Sigma), anti-human Nestin (rabbit IgG, 1∶4,000) , anti-myelin basic protein (MBP) (rat IgG, 1∶500, AbD Serotec, Raleigh, NC, USA), anti-O4 (mouse IgM 1∶1,000, Millipore), and anti-platelet-derived growth factor receptor (PDGFR)α (rabbit IgG, 1∶2,000, Santa Cruz, Santa Cruz, CA, USA).

Techniques: Derivative Assay, Injection, Immunohistochemical staining, In Vivo

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Upregulated expression of miR-4443 and miR-4488 in drug resistant melanomas promotes migratory and invasive phenotypes through downregulation of intermediate filament nestin.

doi: 10.1186/s13046-023-02878-9

Figure Lengend Snippet: Fig. 3 The intermediate filament Nestin is a molecular target of miR-4443 and miR-4488 in melanoma. a Schematic representation of the entire 3’UTR of mRNA relative to nestin containing the two seed regions for miR-4488 and for miR-4443 located at positions 197–203 and 336–342, respectively (source miRWalk3.0). b, c To perform luciferase assays, HEK-293 cells were plated in a 6-wells plate and then co-transfected with a plasmid containing the 3’ UTR of nestin and Renilla luciferase plasmid together with miR-4443 (b), miR-4488 (c) or the relative scrambled (SCR). After 48 h of transfection, cells were harvested and luminescence was evaluated using Dual-Luciferase® Reporter Assay System. The values were calculated as fold change (± SD) compared to SCR considered as 1. d A375 SEN cells were transiently transfected with miR-4443, miR-4488 or with the SCR for 72 h. Cells have been then harvested and lysed to extract total proteins that have been subjected to Western blot analyses with the indicated antibodies (left panel). Tubulin levels have been estimated for the protein equal loading and densitometric analyses were performed using ImageJ software (right panel). Results were expressed as mean values from three independent experiments. The values were calculated as fold change (± SD) compared to SCR considered as 1. e A375 SEN cells transfected as described above have been stained with an antibody PE mouse anti-nestin for 45 min to perform flow cytometry analyses. Non-stained cells were used as negative control. Black arrows indicate the peaks relative to cells transfected with miR-4443 or miR-4488 mimics (left graphs). Quantitative analyses were calculated as “fold change” (± SD) compared to SCR considered as 1 (right graph). Data were analyzed using CytExpert version 2.2 software. f Representative images by confocal microscopy of nestin and DAPI immunostaining of A375 SEN cells transiently transfected as described above. Magnification 63x. All the experiments have been performed at least in triplicate ± standard deviation (SD) and p-value < 0.05 was considered as significant (Student’s t-test)

Article Snippet: The plasmid containing the 3′UTR of human nestin was purchased by Origene.

Techniques: Luciferase, Transfection, Plasmid Preparation, Reporter Assay, Western Blot, Software, Staining, Flow Cytometry, Negative Control, Confocal Microscopy, Immunostaining, Standard Deviation