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: International journal of molecular sciences

Article Title: Effect of 3D Spheroid Culturing on NF-κB Signaling Pathway and Neurogenic Potential in Human Amniotic Fluid Stem Cells.

doi: 10.3390/ijms24043584

Figure Lengend Snippet: Figure 2. Gene expression profile of stemness, metabolic genes, and neuronal markers in control and induced to neural differentiation hAFSCs, grown in 2D and 3D cell cultures. RT-qPCR analysis of hAFSCs (A) stemness-associated genes SOX2, OCT4, NANOG, LIN28A, MSI1, MSI2, and NOTCH1 and cell survival-associated/metabolic genes MYC, TP53, BAK-1, BAX, MCL1, HIF1A, (B) as well as neural differentiation crucial genes MAP2, NCAM1, NSE, NES, TUBB3, NTRK1, BDNF, GFAP, NGF, CNTF, ALDH1L1, GDNF, NEUROD1, VEGF, and VIM. Gene expression analysis was performed using control hAFSCs (not treated, “Ctrl”) and neuronal differentiation-induced hAFSCs (treated for 5 days with 50 ng/mL BDNF + 100 ng/mL NGF + 5 mM KCl + 2 µM RA; denoted as “Neuro”). Control and neuro-induced hAFSCs were cultivated either in 2D or 3D cell cultures. RT-qPCR data are represented as relative fold change over 2D undifferentiated control, normalized for the housekeeping genes GAPDH and RPL13A; values are indicated as mean ± SD (n = 3). Note: * denotes significant difference with p < 0.05, ** denotes significant difference with p < 0.01, as evaluated using one-way repeated-measures ANOVA with Tukey‘s post hoc test.

Article Snippet: For detection of LIN28a, Nestin, and MYC, cells were incubated with primary mouse antibodies against LIN28a (1:200) and Nestin (1:100) or rabbit antibodies against MYC (1:150) (Novus Biologicals, Abingdon, UK) for 1 h, at 37 ◦C, followed by incubation with secondary goat anti-rabbit or goat anti-mouse IgG (H + L) Highly Cross-Adsorbed, Alexa Fluor® 488 antibodies (1:400) (Invitrogen, Thermo Fisher Int.

Techniques: Gene Expression, Control, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Rho GTPases signaling mediates aggressiveness and differentiation in neuroblastoma tumors

doi: 10.1186/s12964-025-02649-3

Figure Lengend Snippet: ARHGAP31 /CdGAP is associated with undifferentiated cells, and its downregulation provokes Cdc42 overexpression. A mRNA expression of ARHGAP31 in adrenergic tumors, mesenchymal tumors or neural crest tissues. Long rank test p value is shown, * = p < 0.05. Dataset: GSE908030. B mRNA expression of ARHGAP31 in SK-N-SH CD44hi-derived undifferentiated secondary tumorspheres (TS2) compared to the bulk cell line. * = p < 0.05, n = 4. C Immunofluorescence showing CdGAP expression in NB48T CD44hi-derived undifferentiated tumorspheres compared to CD44negative-derived ones. D Tumorsphere formation efficiency of SK-N-SH or NB48T PDX-derived cell lines after CdGAP knock down by siRNA or control. * = p < 0.05; *** = p < 0.005. E Expression of the undifferentiated marker nestin in SK-N-SH and NB48T PDX-derived tumorspheres after CdGAP knock down by siRNA or control. ** = p < 0.01; **** = p < 0.001. F Expression of Cdc42 in SK-N-SH and NB48T PDX-derived TS cells after CdGAP knock down by siRNA or control. Representative images from NB48T cells are shown. **** = p < 0.001. G Expression of Sox9 protein in cells from tumorspheres generated from NB48T or sorted CD44hi SK-N-SH and NB48T cells, after CdGAP knock down by siRNA or control. * = p < 0.05; **** = p < 0.001. H Cell viability in CHLA 20 cells, with or without downregulation of CdGAP, over 72 h (** = p < 0.01). I Quantification of proliferating Ki67 expressing cells in NB48T cells with or without downregulation of CdGAP. Graph shows mean ± SEM. Scale bar = 20μm. (* = p < 0.05, n = 3). J Morphological characterization of CHLA 20 and NB48T cells with or without downregulation of CdGAP. Cell segmentation on the cell contour is shown in red. Scale bar = 20 μm. K Immunofluorescence for acetylated tubulin, and DAPI in CHLA 20 cells with or without downregulation of CdGAP. The presence of acetylated tubulin in cell extensions is indicated with red asterisks. Scale bar = 20 μm. L Quantification of the cells positive for acetylated tubulin staining in cellular protrusions after the indicated treatments in CHLA20 cells (mean ± SEM, * = p < 0.05; ** = p < 0.001, n = 3)

Article Snippet: For immunofluorescence, cells were fixed with 4% PFA in PBS for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min at 4 °C and blocked with PBS + 1% BSA for at least 1 h. Primary antibodies used were: CD44 mouse (1:200 BD Pharmingen), Cdc42 rabbit (1:1000 Cell Signaling), Ki67 rabbit (1:200 Thermo Scientific), Nestin rabbit (1:1000 Millipore), Nestin mouse (1:1000 R&D Systems), α-tubulin mouse (1:1000 Sigma), acetylated tubulin mouse (1:1000, Santa Cruz), Tuj1-βIII tubulin mouse (1:1000 abcam) and CdGAP (1:500, Santa Cruz).

Techniques: Over Expression, Expressing, Derivative Assay, Immunofluorescence, Knockdown, Control, Marker, Generated, Staining

Journal: Science advances

Article Title: Significance of birth in the maintenance of quiescent neural stem cells.

doi: 10.1126/sciadv.adn6377

Figure Lengend Snippet: Fig. 2. Preterm birth RG transiently adopt a neurogenic state. (A) Experimental scheme. (B) Representative images of coronal V-SVZ sections stained for Glul RNA (green) at E18.5, full-term P2, and preterm P3. Nuclei were stained with DAPI (blue). (C) Percentages of Glul RNA dots area in the V-SVZ area (n = 3 mice each; one-way ANOVA followed by Tukey-Kramer test). (D) Representative images of coronal V-SVZ sections in GFAP-EGFP mice stained for EGFP (green), Glul (red), and Nestin (white) at E18.5, full-term P2, and preterm P3. Yellow lines, traced outlines of RG based on EGFP staining. (E) Relative expression levels of Glul in RG (each n = 60 cells from three mice; Kruskal-Wallis test followed by Steel-Dwass test). (F) Experimental scheme. (G) Representative projection images of coronal V-SVZ sections stained for pS6 (green), Mash1 (red), and Nestin (white) at 18.5, 21.5, and 26.5 dpc. White arrows, Mash1+pS6−Nestin+RG. Yellow arrows, Mash1−pS6+Nestin+ RG. Blue arrowheads, Mash1+pS6+Nestin+ RG. (H) Densities of Mash1+Nestin+ RG in the V-SVZ at 18.5, 21.5, and 26.5 dpc (n = 4 mice each; unpaired t test). (I) Densities of pS6+Nestin+ RG in the V-SVZ at 18.5, 21.5, and 26.5 dpc (n = 3 mice each; unpaired t test). (J) Percentages of Mash1+ cells in pS6+Nestin+ RG and pS6−Nestin+ RG at 21.5 dpc (n = 4 mice each; Mann-Whitney U test). (K) Density of pS6+Mash1+Nestin+ RG in the V-SVZ at 21.5 dpc (n = 4 mice each; unpaired t test). Dpc, days post coitus; Mife, mifepristone; LV, lateral ventricle. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars, 20 μm [(B), (D), and (G) (single-channel images)] and 50 μm [(G) (merged images)]. Error bars, mean ± SEM. See also fig. S10.

Article Snippet: The following primary antibodies were used: rabbit anti- GS (Glul) (1:500; Abcam), chicken anti- Nestin (1:1000; Aves Labs, Tigard, OR, USA), rabbit anti- Mash1 (1:1000; Abcam), mouse anti- Mash1 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti–phosphoS6 ribosomal protein (pS6) (1:500; Cell Signaling Technology, Beverly, MA, USA), rabbit anti–phospho- p70 S6 kinase (Thr421) (1:500; Thermo Fisher Scientific), guinea pig anti- Dcx (1:500; Millipore), mouse anti- NeuN (1:100; Millipore), rat anti- BrdU (1:100; Abcam), sheep anti- BrdU (1:1000; Fitzgerald, Acton, MA, USA), rat antiGFP (1:500; Nacalai Tesque, Kyoto, Japan), rabbit anti- EGFR (1005) (1:100; Santa Cruz Biotechnology), rabbit anti- DsRed (1:1200; Takara Bio, Kusatsu, Japan), and chicken anti- GFAP (1:1000; Abcam).

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: Stem Cell Research & Therapy

Article Title: Neocortical tissue recovery in severe congenital obstructive hydrocephalus after intraventricular administration of bone marrow-derived mesenchymal stem cells

doi: 10.1186/s13287-020-01626-6

Figure Lengend Snippet: Primary antibodies

Article Snippet: Nestin , BIO-RAD , AHP1739 , RbP , I, FC, VB , 1:100.

Techniques: