Journal: Cancers

Article Title: SLUG Directs the Precursor State of Human Brain Tumor Stem Cells

doi: 10.3390/cancers11111635

Figure Lengend Snippet: Stem-like and progenitor-like brain tumor stem cells (BTSCs) display proneural and mesenchymal transcriptomic profiles respectively, which correlate with epithelial to mesenchymal transition (EMT) and STAT3 pathways’ activity (see also ). ( A ) Schematic representation of BTSC precursor states. ( B ) Bar graph showing our cohort of 57 BTSCs ranked based on the stem-like to progenitor-like score and segregated as stem-like (30 BTSCs) or progenitor-like (27 BTSCs). ( C ) Heatmap representing the expression of proneural and mesenchymal genes (from and listed in ) in stem-like and progenitor-like BTSCs in the same order than B (expression data can be found on the EGA platform). ( D ) Scatter plot showing the correlation between the proneural to mesenchymal and the stem-like to progenitor-like scores in BTSCS. Scatter plots showing the correlation between the proneural to mesenchymal score and ( E ) STAT3 or ( F ) EMT scores in BTSCs segregated as stem-like (blue) or progenitor-like (red). Scatter plot showing ( G ) the correlation of STAT3 and EMT scores and ( H ) the correlation between the proneural to mesenchymal score and E-cadherin expression in BTSCs segregated as stem-like (blue) or progenitor-like (red). All figures are based on RNA-sequencing performed on 57 BTSC lines.

Article Snippet: Wild-type STAT3 (Sino Biological, Wayne, PA, USA) and a constitutively active STAT3 (EF.STAT3C.Ubc.GFP, Addgene, Watertown, MA, USA) were cloned into a constitutive Piggy Back vector (PB513B-1, System Biosciences).

Techniques: Activity Assay, Expressing, RNA Sequencing

Journal: Cancers

Article Title: SLUG Directs the Precursor State of Human Brain Tumor Stem Cells

doi: 10.3390/cancers11111635

Figure Lengend Snippet: SLUG is the key EMT transcription factor in progenitor-like BTSCs (see also ). ( A ) Box and whiskers (min to max) plot of the expression of EMT master regulators in stem-like (blue) and progenitor-like (red) BTSCs. ( B ) Representative western blot showing increased activated STAT3 (pSTAT3-Y705) and higher SLUG levels in progenitor-like (red) compared to stem-like (blue) BTSCs. Quantification relative to loading controls. Scatter plots representing the correlation between SLUG expression and ( C ) STAT3 and ( D ) EMT scores. ( E ) Scatter plots showing the mutually exclusive expression of SLUG with E-cadherin in BTSCs segregated as stem-like (blue) or progenitor-like (red). ( F ) Scatter plot illustrating the inverse correlation between expression of SLUG and available BTSC survival data. ( G ) Scatter plot representing the correlation between STAT3 and EMT scores in SLUG high (red, z-score < 1) and SLUG low (blue, z-score >1) TCGA GBM (glioblastoma) samples (Affymetrix U133a microarray platform). ( H ) Kaplan Meier survival curves from 523 GBM samples segregated around the geometric mean of SLUG expression. 256 samples were below (Low SLUG, in blue, 454 days) and 267 samples above (High SLUG, in red, 386 days) this geometric mean. Expression data is from TCGA Affymetrix U133a microarray platform. Box and whiskers plot in ( A ), scatter plots ( C – E ) and SLUG expression in ( F ) are based on RNA-sequencing performed on 57 BTSC lines.

Article Snippet: Wild-type STAT3 (Sino Biological, Wayne, PA, USA) and a constitutively active STAT3 (EF.STAT3C.Ubc.GFP, Addgene, Watertown, MA, USA) were cloned into a constitutive Piggy Back vector (PB513B-1, System Biosciences).

Techniques: Expressing, Western Blot, Microarray, RNA Sequencing

Journal: Cancers

Article Title: SLUG Directs the Precursor State of Human Brain Tumor Stem Cells

doi: 10.3390/cancers11111635

Figure Lengend Snippet: SLUG is the primary direct transcriptional target of STAT3 in BTSC (see also ). ( A ) Box and whiskers graph (min to max) representing SLUG expression measured by qPCR in 12 BTSCs (as detailed in ) treated with JAK3 inhibitor (R333) and ( B ) representative western blot quantified relative to loading control. ( C ) Representative western blot (with quantification values relative to loading control) showing activated STAT3 (pSTAT3-Y705) and SLUG protein levels in BT67 24 h post epidermal growth factor (EGF), leukemia inibitory factor (LIF), and oncostatin M (OSM) treatments with or without concurrent inhibition of STAT3 with direct STAT3 inhibitor STATTIC (10 µM). ( D ) Bar graph of SLUG expression measured by qPCR in empty vector, wild-type STAT3, or constitutive STAT3, overexpressing BT124. RT-qPCR data representing ( E ) SLUG and ( F ) E-cadherin expression following OSM treatment (10 ng/mL) in BT67. Positive controls (STAT3 and GFAP), negative controls and SLUG enrichment results from ChIP-PCR experiments performed ( G ) on BT67 in growth factor-free media with or without OSM or ( H ) on BT50, BT67, BT69, BT89, BT94, BT147 grown in standard BTSC culture condition (+EGF, +FGF). ( I ) Histograms representing binding of STAT3 on the promoter of SLUG, SNAIL, TWIST, ZEB1, and ZEB2 from whole genome ChIP-seq experiments performed on the same 6 BTSC lines (BT50, BT67, BT69, BT89, BT94, BT147) maintained in standard BTSC culture condition (+EGF, +FGF). ( D – H ), SEM, ns. p>0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Wild-type STAT3 (Sino Biological, Wayne, PA, USA) and a constitutively active STAT3 (EF.STAT3C.Ubc.GFP, Addgene, Watertown, MA, USA) were cloned into a constitutive Piggy Back vector (PB513B-1, System Biosciences).

Techniques: Expressing, Western Blot, Control, Inhibition, Plasmid Preparation, Quantitative RT-PCR, Binding Assay, ChIP-sequencing

Journal: Cancers

Article Title: SLUG Directs the Precursor State of Human Brain Tumor Stem Cells

doi: 10.3390/cancers11111635

Figure Lengend Snippet: SLUG promotes migration and invasion both in vitro and in vivo (see also ). Bar graphs representing ( A ) SLUG and ( B ) E-cadherin expression in BT69 control (CTRL) and overexpressing SLUG (SLUG_OE) as measured by RT-PCR (* p < 0.05; ** p < 0.01). ( C ) Representative images of cells on the upper side of the membrane (highlighted in yellow) and cells that have migrated to the bottom side (highlighted in red) and ( D ) quantified migration of CTRL and SLUG_OE cells. ( E ) Graph of STAT3 inhibition (STATTIC) impact on migration of BT69 CTRL and SLUG_OE. ( F ) Representative overlaid images of spheres embedded in collagen at T0 (highlighted in yellow) and pictures of invaded cells at end point (highlighted in red). ( G ) Graphical representation of the quantified invasion of BT69 CTRL and SLUG_OE cells in vitro (area of invaded cells (highlighted in red) was normalized to the area of the imbedded spheres at T0 (highlighted in yellow). ( H ) Representative pictures of the invasive behavior of BT69 CTRL and SLUG_OE cells in a corpus callosum implantation assay. hNucl positive cells were overlaid with large red dots on the high resolution images to illustrate the tumor cell distribution throughout the brain at endpoint. ( I ) Total hNucl positive area throughout the brain sections. ( J ) Graphical representation of the hNucl positive area in invaded zones (represented as rectangles) normalized to total hNucl positive area. Error bars represent SEM ( A – J ).

Article Snippet: Wild-type STAT3 (Sino Biological, Wayne, PA, USA) and a constitutively active STAT3 (EF.STAT3C.Ubc.GFP, Addgene, Watertown, MA, USA) were cloned into a constitutive Piggy Back vector (PB513B-1, System Biosciences).

Techniques: Migration, In Vitro, In Vivo, Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Membrane, Inhibition

Journal: Cancers

Article Title: SLUG Directs the Precursor State of Human Brain Tumor Stem Cells

doi: 10.3390/cancers11111635

Figure Lengend Snippet: SLUG-induced transcriptional changes are associated with progenitor-like BTSC precursor state and recurrence (see also ). ( A ) Revigo visualization of Gene Ontology analysis from RNA sequencing performed on BT50 CTRL and SLUG_OE cells. ( B ) Heatmap of the 180 differentially expressed genes between BT50 CTRL and SLUG_OE cells in the 57 BTSCs and bar graphs of their respective SLUG_OE, stem-like to progenitor-like (SL/PL), STAT3, and EMT scores (asterisk mark BTSCs from recurrent primary GBMs). ( C ) Box and whiskers plot (min to max) representing the SLUG_OE score of BTSCs that were derived from primary GBMs compared to those derived from recurrent GBMs (exclusively from de novo GBMs). ( D ) Scatter plot of SLUG_OE score versus proneural to mesenchymal score.

Article Snippet: Wild-type STAT3 (Sino Biological, Wayne, PA, USA) and a constitutively active STAT3 (EF.STAT3C.Ubc.GFP, Addgene, Watertown, MA, USA) were cloned into a constitutive Piggy Back vector (PB513B-1, System Biosciences).

Techniques: RNA Sequencing, Derivative Assay

Journal: Cancer cell

Article Title: The SS18-SSX Fusion Oncoprotein Hijacks BAF Complex Targeting and Function to Drive Synovial Sarcoma

doi: 10.1016/j.ccell.2018.05.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Anti-V5 tag (D3H8Q) (ChIP) , Cell Signaling Technology , Cat#13202; RRID:AB_2687461.

Techniques: Microarray, Recombinant, Sample Prep, Sequencing, shRNA, Control, CRISPR, Plasmid Preparation, Software

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Pathway analysis of microarray data on genes differentially expressed between control and SH-SY5Y-APP cells. b ChIP-seq screening results in SH-SY5Y cells showed that the region of AICD interaction is in the fourth intron of the JNK3 locus in chromosome 4. TSS, transcription starting site. c Validation of the ChIP-seq results. SH-SY5Y cells were transfected with either pcDNA4/V5-His or pcDNA4/V5-His-hAICD59 plasmid, and subjected to ChIP assay, using normal mouse IgG as control. Input, 5% of the sonicated chromatin. Left panel, real-time PCR results show V5, but not IgG control interacts with the JNK3 intron region ( n = 3). Right panel, representative image shows the real-time PCR products resolved by agarose gel. The molecular weight of the DNA ladder shown in bp on the left. d Real-time PCR results showing the JNK3 mRNA level in pcDNA4/V5-His (control) or pcDNA4/V5-His-hAICD59 (AICD) transfected SH-SY5Y cells ( n = 3). Student’s t- test: ** p < 0.01. Error bars represent SEM. e Luciferase assay showing the enhancer activity of the AICD-interacting site. One-way ANOVA followed by post-hoc Bonferroni test: ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Microarray, Control, ChIP-sequencing, Biomarker Discovery, Transfection, Plasmid Preparation, Sonication, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Luciferase, Activity Assay

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Intact retinae from wild-type mice were fixed, paraffin-embedded and 5 μm thick longitudinal sections were cut. The sections were stained with anti-JNK3, anti-APP and anti-Tuj1 antibodies after antigen retrieval. The inserts show lower magnification photomicrographs illustrating the immunofluorescence signal distribution across all the cell layers of the retina. APP and JNK3 were co-expressed in Tuj1 + RGCs. b Intact retinae from wild-type, APP-null or JNK3-null mice were stained with anti-APP or anti-JNK3 antibodies after antigen retrieval. The inserts show lower magnification photomicrographs illustrating the immunofluorescence signal distribution across all the cell layers of the retina. The anti-APP and anti-JNK3 antibodies were specific as no immunofluorescence signal is seen in the APP-null and JNK3-null retinal sections, respectively. c Adult, wild-type mouse retinae were retrogradely labeled with OHSt, fixed, and 5 μm thick sections were cut. The sections were stained with anti-APP and anti-JNK3 antibodies without antigen retrieval as antigen retrieval destroys the OHSt signal. Arrow heads indicate several OHSt-labeled RGCs, which co-express both APP and JNK3. Scale bar, 20 μm

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Staining, Immunofluorescence, Labeling

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Quantitative analysis of relative mRNA levels of Jnk s in intact retinae or retinae 1 day (1 d), 1 week (1 w) or 2 weeks (2 w) after ONA ( n = 3 for each time point). b Representative western blots from at least three biological repeats showing JNK expression after ONA. Intact retina or retinae 1 d, 1 w, 2 w after ONA were harvested, lysed and immunoblotted using antibodies against JNK1, JNK2, JNK3, pJNK or GAPDH. The location of molecular weight markers is shown in kDa on the right. c Representative fluorescence microscope images show immunostaining of mouse retina flat-mounts from wild-type or JNK1, JNK2, or JNK3-null mice ( n = 6 for each genotype) 14 days after ONA using an anti-Tuj1 antibody. d The bar chart shows quantitative analysis of Tuj1 + RGC numbers 14 days after ONA. Scale bar, 50 μm. One-way ANOVA followed by post-hoc Bonferroni test: ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Western Blot, Expressing, Molecular Weight, Fluorescence, Microscopy, Immunostaining

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: Representative western blots show JNK3 expression in a pcDNA3-FLAG-APP695 transfected or pcDNA3 transfected SH-SY5Y cells, e retinae from wild-type or APP-null mice, or i pcDNA4/V5-His-AICD59 or pcDNA4/V5-His transfected SH-SY5Y cells. The samples were lysed, resolved in SDS-page gel, and immunoblotted using antibodies against JNK3 or GAPDH. b , f , j The bar charts show quantitative analysis of relative JNK3 expression ( n = 3 for each experiment). Representative western blots show phosphorylative activation of JNK3 in c APP695-overexpressing or normal SH-SY5Y cells, g retinae from wild-type or APP-null mice, or k pcDNA4/V5-His-AICD59 or pcDNA4/V5-His transfected SH-SY5Y cells. JNK3 was immunoprecipitated with anti-JNK3 antibody and then detected for kinase activity with anti-phospho-JNK (pJNK) or anti-JNK3 antibody. Whole-cell lysate western blots show the downstream substrate pc-Jun73 or c-Jun level. d , h , l The bar charts show quantitative analysis of activated pJNK/mg lyate proteins or pc-Jun73/c-Jun ratio ( n = 3 for each experiment). Student’s t -test: * p < 0.05; ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Western Blot, Expressing, Transfection, SDS Page, Activation Assay, Immunoprecipitation, Activity Assay

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a Representative western blots show JNK3 expression and overall phosphorylative activation of JNK in retinae from wild-type or APP-null mice 1 day (1 d), 1 week (1 w) and 2 weeks (2 w) after ONA ( n = 3 for each time point). Anti-JNK3, anti-pJNK and anti-GAPDH antibodies were used to detect the respective protein expression. b The bar charts show quantitative analysis of relative JNK3 expression or overall phosphorylative activation of JNK in the ipsilateral-injured retinae vs. the contralateral-uninjured retina. One-way ANOVA followed by post-hoc Bonferroni test: ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Western Blot, Expressing, Activation Assay

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: a – d Representative western blots show JNK3 expression and overall phosphorylative activation of JNK in SH-SY5Y-APP cells treated with vehicle or γ-secretase inhibitors a L-685,458 (5 μM) or c BMS 299897 (1 μM). b , d The bar charts show quantitative analysis of relative JNK3 expression and overall phosphorylative activation of JNK normalized to the GAPDH ( n = 3 for each treatment). e Representative western blots show phosphorylative activation of JNK3 in retinae from vehicle-treated or γ-secretase inhibitor BMS 299897 treated mice 2 weeks after ONA. f Representative fluorescence microscope images show immunostaining of mouse retina flat-mounts from vehicle or γ-secretase inhibitor BMS 299897 treated mice 2 weeks after ONA using anti-Tuj1 antibody. Scale bar, 50 μm. g The bar chart shows quantitative analysis of the surviving Tuj1 + RGC number in injured mouse retinae 14 days after ONA ( n = 4 for each treatment). Student’s t -test: ** p < 0.01. Error bars represent SEM

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Western Blot, Expressing, Activation Assay, Fluorescence, Microscopy, Immunostaining

Journal: Cell Death and Differentiation

Article Title: APP upregulation contributes to retinal ganglion cell degeneration via JNK3

doi: 10.1038/s41418-017-0005-3

Figure Lengend Snippet: ONA induces upregulation of APP expression. APP is sequentially cleaved by α- or β-secretase and γ-secretase, producing several fragments and AICD, which is inhibited by APP deficiency or BMS 299897. AICD is translocated to the nucleus and upregulates JNK3 expression (probably functioning with other partners). Phosphorylated JNK3 then contributes to RGC death

Article Snippet: Rabbit antibodies against APP (Y188, Abcam) [ ], JNK1 (catalog no. MAB17761, R&D Systems), JNK2 (catalog no. MAB1846, R&D Systems) [ ], JNK3 (catalog no. 2305, Cell Signaling Technology), p-cJun73 (catalog no. 9164, Cell Signaling Technology) and c-Jun (catalog no. 9165, Cell Signaling Technology); mouse antibodies against GAPDH (catalog no. ab8245, Abcam) [ ], V5 (catalog no. R96025, Invitrogen), pJNK (catalog no. 9255, Cell Signaling Technology) [ ] and JNK3 (catalog no. CP10162, Cell Applications) were used.

Techniques: Expressing