Journal: Cell

Article Title: SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses

doi: 10.1016/j.cell.2020.10.004

Figure Lengend Snippet:

Article Snippet: At 72 hours post infection, the samples were harvested and the resulting whole cell lysates were probed by western blot with either sheep anti-NSP or mouse anti-actin (Developmental Studies Hybridoma Bank JLA20, antibody registry ID: AB_528068) primary antibodies.

Techniques: Virus, Recombinant, Transfection, RNA Sequencing, Software

Journal: iScience

Article Title: The Taspase1/Myosin1f-axis regulates filopodia dynamics

doi: 10.1016/j.isci.2022.104355

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-RNA Polymerase II/POLR2A , Novus Biologicals , Cat#: NB200-598; RRID: AB_2167465.

Techniques: Recombinant, Western Blot, Migration, Isolation, Amplification, Plasmid Preparation, Software

Journal: Translational Psychiatry

Article Title: Elevation of Il6 is associated with disturbed let-7 biogenesis in a genetic model of depression

doi: 10.1038/tp.2016.136

Figure Lengend Snippet: Gene and miRNA expression levels were measured in the prefrontal cortex of naïve FSL and FRL rats using RT-PCR. ( a ) Increased Il6 and LIN28B, and decreased DROSHA levels in the FSL rats compared to FRL. ( b ) FSL rats exhibited decreased expression of several let-7 family members; Levels of let-7b, let-7c, let-7f, let-7i and miR-98 were significantly reduced. ( c ) RNA immunoprecipitation showed an enrichment of primary let-7 transcripts pri-let-7c-1, pri-let-7i and pri-mir-98 bound to LIN28B in FSL. ( d ) The reduction of mature let-7 was not associated with primary let-7 transcripts changes. Gene expression data are presented as relative quantifications (R.Q.), two reference genes ( Gapdh and Ppia ) were used for normalization of Il6 , Lin28b , Drosha and the primary miRNA genes. Rnu5g was used for normalization of mature miRNAs. Lower panels in a (right) show representative western blot images of LIN28B and DROSHA from the same gel, respectively with β-actin as loading control. RIP data are presented as relative value to FRL. Data are presented as group means±s.e.m. For all figures: n =5–7 animals per group, * P <0.05, ** P <0.01, *** P <0.001. FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line; miRNA, microRNA; RIP, RNA immunoprecipitation; RT-PCR, real-time polymerase chain reaction.

Article Snippet: Immunoblotting was performed overnight at 4 °C with a polyclonal rabbit anti-LIN28B antibody (1:500 dilution; #5422, Cell Signaling Technology, Danvers, MA, USA) or with a polyclonal goat anti-DROSHA antibody (1:1000 dilution; ab58589; Abcam plc, Cambridge, UK) separately, and with a mouse monoclonal anti-β-actin antibody (1:10 000; A5316; Sigma-Aldrich, St Louis, MO, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, RNA Immunoprecipitation, Gene Expression, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Translational Psychiatry

Article Title: Elevation of Il6 is associated with disturbed let-7 biogenesis in a genetic model of depression

doi: 10.1038/tp.2016.136

Figure Lengend Snippet: Gene and miRNA expression levels were measured in the prefrontal cortex of FSL with/without access to running wheel (FSL-runners versus FSL-controls) using RT-PCR. ( a ) Physical activity normalized Il6 levels in the FSL-runner group but had no effect on Lin28b and Drosha mRNA levels. ( b ) In line with an Il6 reduction in the FSL rats that were running, let-7i and miR-98 showed significantly increased expression. Specifically, upregulation of let-7i was associated with primary let-7i overexpression ( c ). ( d ) Chromatin immunoprecipitation (ChIP) showed an increased histone H4 acetylation (H4ac) but not histone H3 acetylation (H3ac) within the pri-let-7i promoter of the FSL-runners. Gene expression data were presented as relative quantifications (R.Q.), two reference genes ( Gapdh and Ppia ) were used for normalization of Il6 , Lin28b , Drosha and the primary miRNA genes. Rnu5g was used for normalization of mature miRNAs. ChIP data are presented as percentage of genomic input DNA. Data are presented as group means±s.e.m. For all figures: n =5–7 animals per group, * P <0.05, ** P <0.01, *** P <0.001. FSL, Flinders Sensitive Line; mRNA, messenger RNA; miRNA, microRNA; RT-PCR, real-time polymerase chain reaction.

Article Snippet: Immunoblotting was performed overnight at 4 °C with a polyclonal rabbit anti-LIN28B antibody (1:500 dilution; #5422, Cell Signaling Technology, Danvers, MA, USA) or with a polyclonal goat anti-DROSHA antibody (1:1000 dilution; ab58589; Abcam plc, Cambridge, UK) separately, and with a mouse monoclonal anti-β-actin antibody (1:10 000; A5316; Sigma-Aldrich, St Louis, MO, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Over Expression, Chromatin Immunoprecipitation, Gene Expression, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation *

doi: 10.1074/jbc.M114.622274

Figure Lengend Snippet: LH3 provides a cell surface docking mechanism for MMP-9 by recognizing its FN domain. A, mass spectrometry analysis. PLOD3_HUMAN (LH3) appeared to be specifically pulled down by MMP-9 and the FN domain according to mass spectrometry analysis with 95% probability (140% probability variance). Coomassie Blue staining of the pulldown of labeled MMP-9, FN, and ΔFN is shown (right panel). B, MMP-9 interacts with endogenous LH3 at the fibroblast cell surface via its FN domain. A representative anti-LH3 antibody immunoblot of anti-v5 antibody immunoprecipitates shows that endogenous LH3 is immunoprecipitated with both v5-tagged MMP-9 and its recombinant FN domain (v5 IP MMP-9 and FN), confirming the specificity of the interaction via fibronectin type II-like motifs. Anti-FLAG antibody immunoprecipitations (IP) constitute a control. C, interaction between MMP-9 and LH3 decreases upon LH3 depletion. PLA analysis between v5-tagged MMP-9 or ΔFN and endogenous LH3 in HSF showing specificity of the interaction between LH3 and the FN domain of MMP-9 (left panel) and impairment of the interaction when LH3 is depleted (KD) (right panel). nb, number. Results represent mean values ±S.E. (error bars). *, p ≤ 0.05.

Article Snippet: Chemical compounds used included:4-aminophenylmercuric acetate (164610, Calbiochem), Complete Mini EDTA-free protease inhibitors (11836170001, Roche Applied Science), FcR blocking reagent (130-059-901, Miltenyi Biotec), FuGENE 6 Transfection Reagent (E2692, Promega), Interferin (409-01, Polyplus Transfection), Sulfo-SBED Biotin Label Transfer Reagent (33034, Pierce), SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Scientific Pierce), human TGF-β1 (100-B-001, R&D Systems), Duolink II PLA Probe Anti-Mouse PLUS (DUO92001, Sigma-Aldrich), Duolink II PLA Probe Anti-Rabbit MINUS (DUO92005, Sigma-Aldrich), Duolink In Situ Detection Reagents Red (DUO92008, Sigma-Aldrich), and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) (human; three unique 27-mer siRNA duplexes) (SR305927, Origene).

Techniques: Mass Spectrometry, Staining, Labeling, Western Blot, Immunoprecipitation, Recombinant

Journal: PLoS ONE

Article Title: The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification

doi: 10.1371/journal.pone.0214110

Figure Lengend Snippet: (A) USP9X interaction network showing all high confidence binding partners connected by solid lines (Saint > 0.9, FDR < 0.05, Avg. Psms > 10). Dotted lines indicate interactions reported in the BioPlex network [ , ]. Results are representative of 3 independent experiments. (B) Western blots of Peg10 3xf/+ Usp9X fl/y Rosa26 .CreER T2 ESCs after immunoprecipitation (IP) of USP9X. Epitope-tagged PEG10 was detected with anti-FLAG antibody. Where indicated, ESCs were treated with 4-hydroxytamoxifen (4-OHT) to delete Usp9x . Results are representative of 2 independent experiments. (C) Western blots of ESCs. (D) Geyser plot showing proteins differentially ubiquitinated in Usp9x C1566A/y ESCs versus control Usp9x +/y ESCs (p < 0.05, log 2 fold change > 2). Results are representative of 2 independent experiments. (E) Line plot showing the fold-change in abundance of all ubiquitinated PEG10 peptides between Usp9x +/y and Usp9x C1566A/y ESCs. Each grey line corresponds to a unique KGG peptide spectral match peptide. The red line indicates a model-based protein abundance estimate. AUC, area under the curve. (F) Diagram indicating the position of the USP9X-regulated ubiquitination (Ub) sites in PEG10. Triangles depict ubiquitination sites identified only in Usp9x C1566A/y ESCs. Circles depict sites found in Usp9x +/y and Usp9x C1566A/y ESCs. Circle size indicates the extent to which ubiquitination was increased by USP9X inactivation. (G) Western blots of Peg10 3xf/+ Usp9x fl/y Rosa26 .CreER T2 ESCs. Where indicated, cells were treated with 10 μM MG-132 for 4 h. Results are representative of 2 independent experiments.

Article Snippet: In brief, 2x10 7 TSCs were UV (254 nm)-crosslinked at 0.4 J/cm 2 , snap frozen, and then lysed prior to treatment with RNase I. PEG10 was immunoprecipitated with anti-PEG10 antibody pre-coupled to sheep anti-mouse Dynabeads (Thermo Fisher) overnight at 4°C.

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Control, Quantitative Proteomics, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification

doi: 10.1371/journal.pone.0214110

Figure Lengend Snippet: (A) Western blots of HEK293T cells or extracellular vesicles recovered from the culture medium after transfection with VSV-G and increasing amounts of Gag-Pol cDNA. Results are representative of 2 independent experiments. (B) Western blots of extracellular vesicles in panel A after sucrose gradient density centrifugation. The upper blot shows cells transfected with HIV-1 Gag, whereas the lower blot shows cells transfected with PEG10-RF1. We speculate that the faster migrating PEG10-RF1 species is a processed form of the protein. Results are representative of 2 independent experiments. (C) Western blots of VLPs. Results are representative of 3 independent experiments. (D) Electron micrographs of negatively stained VLPs. Scale bar, 20 nm. Results are representative of 2 independent experiments. (E) Western blots of wild-type ESCs and TSCs. Results are representative of 2 independent experiments. (F) Western blots of HEK293T cells ectopically expressing wild-type (WT) mouse PEG10 or a PEG10 frameshift (FS) mutant that can only make PEG10-RF1/2. Results are representative of 2 independent experiments. (G) Western blots of extracellular vesicles shed from WT or PEG10-deficient (KO) TSCs and enriched by differential centrifugation. Results are representative of 5 independent experiments. (H) Western blots of extracellular vesicles shed from WT TSCs and analyzed by sucrose gradient density centrifugation. Results are representative of 2 independent experiments. (I) Electron micrographs of TSC extracellular vesicles after immuno-gold labeling for PEG10. Scale bar, 50 nm.

Article Snippet: In brief, 2x10 7 TSCs were UV (254 nm)-crosslinked at 0.4 J/cm 2 , snap frozen, and then lysed prior to treatment with RNase I. PEG10 was immunoprecipitated with anti-PEG10 antibody pre-coupled to sheep anti-mouse Dynabeads (Thermo Fisher) overnight at 4°C.

Techniques: Western Blot, Transfection, Centrifugation, Staining, Expressing, Mutagenesis, Labeling

Journal: PLoS ONE

Article Title: The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification

doi: 10.1371/journal.pone.0214110

Figure Lengend Snippet: (A) PEG10 interaction network showing all high confidence binding partners connected by solid lines (Saint > 0.9, FDR < 0.05, Avg. Psms > 10). Dotted lines indicate interactions reported in the Bioplex interaction network. Proteins co-immunoprecipitated from both TSCs and ESCs are colored red, and those unique to ESCs are green. Results are representative of 3 independent experiments. (B) Western blots before and after immunoprecipitation (IP) of 3xFLAG.PEG10 from knock-in ESCs, or as a control, PEG10-deficient (KO) ESCs. Results are representative of 2 independent experiments.

Article Snippet: In brief, 2x10 7 TSCs were UV (254 nm)-crosslinked at 0.4 J/cm 2 , snap frozen, and then lysed prior to treatment with RNase I. PEG10 was immunoprecipitated with anti-PEG10 antibody pre-coupled to sheep anti-mouse Dynabeads (Thermo Fisher) overnight at 4°C.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Knock-In, Control

Journal: PLoS ONE

Article Title: The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification

doi: 10.1371/journal.pone.0214110

Figure Lengend Snippet: (A) Western blots of C57BL/6 mouse tissues. Results are representative of 3 independent experiments. (B) Mouse tissue sections immunolabeled for PEG10 (brown). AC, adrenal cortex. AM, adrenal medulla. LN, Labyrinth. T, Trophoblast. DB, decidua basalis. ST, Sertoli cells. LC, Leydig cells. PN, pars nervosa. PI, pars intermedia. PD, pars distalis. Th, thalamus. Hyp, Hypothalamus. Scale bar, 100 μm. Results are representative of 3 independent experiments. (C) Immunofluorescence staining of PEG10 in ESCs or TSCs. Scale bar, 25 μm. Results are representative of 2 independent experiments. (D) Western blots of ESCs and TSCs that were fractionated into cytoplasmic (cyto) and nuclear (nuc) compartments. Results are representative of 3 independent experiments.

Article Snippet: In brief, 2x10 7 TSCs were UV (254 nm)-crosslinked at 0.4 J/cm 2 , snap frozen, and then lysed prior to treatment with RNase I. PEG10 was immunoprecipitated with anti-PEG10 antibody pre-coupled to sheep anti-mouse Dynabeads (Thermo Fisher) overnight at 4°C.

Techniques: Western Blot, Immunolabeling, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification

doi: 10.1371/journal.pone.0214110

Figure Lengend Snippet: (A) Western blots of TSCs differentiated in 20% oxygen for the times indicated. Peg10 mRNA expression was determined by quantitative RT-PCR. (B) Micrographs of wild-type (WT) and PEG10-deficient (KO) TSCs. Results are representative of 5 independent experiments. (C) Principal Component Analysis (PCA) of TSC RNA-seq datasets using log2 RPKM values.

Article Snippet: In brief, 2x10 7 TSCs were UV (254 nm)-crosslinked at 0.4 J/cm 2 , snap frozen, and then lysed prior to treatment with RNase I. PEG10 was immunoprecipitated with anti-PEG10 antibody pre-coupled to sheep anti-mouse Dynabeads (Thermo Fisher) overnight at 4°C.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, RNA Sequencing

Journal: PLoS ONE

Article Title: The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification

doi: 10.1371/journal.pone.0214110

Figure Lengend Snippet: (A and B) Volcano plots of total protein levels (A) or phosphorylation levels at unique phosphosites (B) in wild-type (WT) versus PEG10-deficient (KO) TSCs after 0 and 5 days of differentiation. Results are representative of 3 independent experiments. (C) Venn diagram indicates the total number of proteins identified and quantified by global proteome profiling (GPP) and phosphoproteome profiling (pSTY). (D) Fraction of proteins (GPP) or phosphosites on Ser/Thr/Try (pSTY) with levels changing more than 2-fold (p-value < = 0.05) in PEG10 KO TSCs compared to WT TSCs after 0 and 5 days of differentiation. (E) Western blots of WT and PEG10 KO TSCs after 0 and 9 days of differentiation. (F) Pathways highlighted by the Broad Institute gene set enrichment analysis of proteins with significantly changing phosphorylation levels in PEG10 KO TSCs compared to WT TSCs. (G) Diagram indicates the position of phosphosites in PEG10.

Article Snippet: In brief, 2x10 7 TSCs were UV (254 nm)-crosslinked at 0.4 J/cm 2 , snap frozen, and then lysed prior to treatment with RNase I. PEG10 was immunoprecipitated with anti-PEG10 antibody pre-coupled to sheep anti-mouse Dynabeads (Thermo Fisher) overnight at 4°C.

Techniques: Phospho-proteomics, Western Blot

Journal: PLoS ONE

Article Title: The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification

doi: 10.1371/journal.pone.0214110

Figure Lengend Snippet: (A) Venn diagram showing RNA transcripts bound to PEG10 after 0 and 5 days of differentiation under normoxic conditions. (B) Graph indicates the distribution of 5'-end eCLIP reads after immunoprecipitating PEG10 from wild-type (WT) and PEG10-deficient (KO) TSCs. Reads are normalized to sequencing depth. (C) Diagram indicates the percentage distribution of nucleotides in PEG10-bound and unbound 3' UTRs. (D) eCLIP-seq data from WT and PEG10 KO TSCs. Blue boxes at the top indicate the location of the genes. (E) Scatter plot comparing RNA-seq and eCLIP peak scores from WT and PEG10 KO TSCs. Genes are shaded red if they are up-regulated in WT TSCs by RNA-seq (log2 fold change >1 and p-value <0.05), grey if they are unchanged (log2 fold change <1 or >-1), and green if they are down-regulated in WT TSCs (log2 fold change <-1 and p-value <0.05). Results are representative of 2 independent experiments.

Article Snippet: In brief, 2x10 7 TSCs were UV (254 nm)-crosslinked at 0.4 J/cm 2 , snap frozen, and then lysed prior to treatment with RNase I. PEG10 was immunoprecipitated with anti-PEG10 antibody pre-coupled to sheep anti-mouse Dynabeads (Thermo Fisher) overnight at 4°C.

Techniques: Sequencing, RNA Sequencing

Journal: Communications Biology

Article Title: Plexin-B2 facilitates glioblastoma infiltration by modulating cell biomechanics

doi: 10.1038/s42003-021-01667-4

Figure Lengend Snippet: a Diagram illustrating structure of Plexin-B2 precursor and mature form (during maturation, Plexin-B2 is cleaved into a non-covalently linked complex of α and β chains). WB with an antibody against the extracellular domain of Plexin-B2 shows a robust expression of Plexin-B2 in SD2 and SD3 GSCs. Note that cells typically express both precursor and mature forms of Plexin-B2, hence the double band pattern. b IF images of cultured GSCs demonstrate the absence of surface expression of Plexin-B2 (PB2) in cells with CRISPR KO. IF images of cells stained with isotype IgG control are shown in the bottom panels. c IF images of coronal brain sections with SD2 GSC transplants at 147 days post injection (dpi). Note the diffuse infiltration of tumor cells (hum. nuc. Ag + ) in striatum and corpus callosum (CC) (arrows) in the control transplant, while PB2-KO GBM cells were mainly confined near the injection site. Also note tumor cell aggregation in collective migration streams at the tumor periphery in PB2-KO transplant (arrowheads), in contrast to the diffuse invasion pattern in control transplant. Quantifications on the right show the relative density of GBM cells (normalized to tumor core) in rings of increasing radius from the tumor core. n = 3 mice per genotype. Two-way ANOVA, *** P < 0.001. d IF images of the CC region show abundant tumor cells (hum. nuc. Ag + ) crossing midline (dotted line) in control transplant at 209 dpi, but much fewer detectable tumor cells in contralateral CC in PB2-KO transplant. Quantifications show the relative abundance of GBM cells found in segments of 0.3 mm increment in contralateral CC. n = 4 mice per genotype. Two-way ANOVA, ** P < 0.01. e Kaplan–Meier survival curves of mice transplanted with control or PB2-KO SD2 GSCs. No mice died for up to 209 dpi for either cohort ( n = 7 mice per cohort), reflecting slow tumor expansion of SD2 GSCs in vivo. f IF images of coronal brain sections from mice transplanted with SD3 GSCs at 29 dpi. GBM cells with PB2-KO were more restricted in their infiltration than control cells. Quantifications on the right show the relative density of GBM cells (normalized to tumor core) in rings of increasing radius from the tumor core. n = 3 mice per genotype. Two-way ANOVA, *** P < 0.001. g Enlarged images of contralateral CC are shown for SD3 transplant. Midline is denoted by a dotted line. Quantifications show the relative abundance of GBM cells found in segments of 0.3 mm increment in contralateral CC. n = 3 mice per genotype. Two-way ANOVA, * P < 0.05. h Kaplan–Meier survival analysis shows that mice bearing intracranial transplants of PB2-KO SD3 GSCs survived significantly longer than mice with transplants of control GSCs: n = 6 mice per cohort; median survival 74.5 days vs. 49.5 days; * P = 0.014, Log-rank test.

Article Snippet: Antibodies used for western blot analysis (WB) or immunofluorescence (IF) were as follows: anti-β-actin, Sigma A1978 (host species: mouse), dilution 1:10,000 for WB anti-EGFR, Cell Signaling Technology 4267 (rabbit), 1:1000 for WB anti-human nuclear antigen (HNA), Millipore MAB1281 (mouse), 1:250 for IF anti-integrin β1 (human-specific clone TS2/16), Santa Cruz sc-53711 (mouse), 1:300 for IF anti-laminin, Millipore MAB1905 (rat), 1:200 for IF anti-MBP, CST 78896 (rabbit), 1:100 for IF anti-MET, Cell Signaling Technology 8198 (rabbit), 1:1000 for WB anti-pMLC2 (Ser19), Cell Signaling Technology 3671 (rabbit), 1:100 for IF anti-PDGFRα, Cell Signaling Technology 3174 (rabbit), 1:1000 for WB anti-PECAM-1/CD31, BD Biosciences 553370 (rat), 1:300 for IF anti-Plexin-B1, R&D systems AF3749 (goat), 1:400 for WB anti-Plexin-B2, extracellular domain, R&D systems AF5329 (sheep), 1:400 for WB, 1:100 for IF anti-Plexin-B2, intracellular domain, Abcam ab193355 (rabbit), 1:1000 for WB anti-Plexin-B3, R&D systems AF4958 (sheep), 1:400 for WB anti-Sema4B, Proteintech 16934-1-AP (rabbit), 1:400 for WB anti-Sema4C, LSBio C150056 (sheep), 1:400 for WB anti-YAP/TAZ, Santa Cruz sc101199 (mouse), 1:100 for IF.

Techniques: Expressing, Cell Culture, CRISPR, Staining, Injection, Migration, In Vivo

Journal: Communications Biology

Article Title: Plexin-B2 facilitates glioblastoma infiltration by modulating cell biomechanics

doi: 10.1038/s42003-021-01667-4

Figure Lengend Snippet: a Left, diagram of cell dispersion assay with 3D aggregates plated on the laminin-coated surface and analyzed after 4 h for dispersion of cell from aggregates. Center, micrographs of DAPI stained aggregates and surrounding dispersed cells (arrows). Right, quantifications of cell dispersion, normalized to mean of the control condition. Box plots and whiskers indicate quartiles, center lines indicate median. n = 18–34 spheres per group; one-way ANOVA with Dunnett’s multiple comparison test of each group against control; * P < 0.05, ** P < 0.01, *** P < 0.001. b Diagram illustrates the hanging drop cell aggregation assay. Photos of drops show aggregates formed by SD2 GSCs with the indicated Plexin-B2 manipulation, 96 h after seeding. Right, quantifications of aggregate numbers and sizes at 24 and 96 h. n = 5–11 hanging drops per group; one-way ANOVA with Dunnett’s multiple comparison test of each group against control; * P < 0.05, *** P < 0.001. c Left, diagram of differential hanging drop cell aggregation assay with GSCs of different genotypes labeled with green or red CellTracker dyes, and mixed 1:1 before seeding. Right, fluorescence images of SD2 aggregates after 24 h, revealing that PB2-KO cells congregated in the center (arrows), while control cells were mainly at the periphery, illustrating stronger adhesiveness between PB2-KO cells. The mixture of GSCs with identical genotypes (Ctrl + Ctrl, or KO + KO) leads to evenly distributed aggregates. d Fluorescence images of SD2 GSCs expressing mCherry that had been embedded as aggregates in 3D fibrin gel matrix. Note the striking differences in the growth/invasion patterns after 27 days: control cells invaded diffusively as individual cells (arrowheads), whereas PB2-KO GSCs invaded the surrounding matrix collectively as fasciculated migration streams (arrows).

Article Snippet: Antibodies used for western blot analysis (WB) or immunofluorescence (IF) were as follows: anti-β-actin, Sigma A1978 (host species: mouse), dilution 1:10,000 for WB anti-EGFR, Cell Signaling Technology 4267 (rabbit), 1:1000 for WB anti-human nuclear antigen (HNA), Millipore MAB1281 (mouse), 1:250 for IF anti-integrin β1 (human-specific clone TS2/16), Santa Cruz sc-53711 (mouse), 1:300 for IF anti-laminin, Millipore MAB1905 (rat), 1:200 for IF anti-MBP, CST 78896 (rabbit), 1:100 for IF anti-MET, Cell Signaling Technology 8198 (rabbit), 1:1000 for WB anti-pMLC2 (Ser19), Cell Signaling Technology 3671 (rabbit), 1:100 for IF anti-PDGFRα, Cell Signaling Technology 3174 (rabbit), 1:1000 for WB anti-PECAM-1/CD31, BD Biosciences 553370 (rat), 1:300 for IF anti-Plexin-B1, R&D systems AF3749 (goat), 1:400 for WB anti-Plexin-B2, extracellular domain, R&D systems AF5329 (sheep), 1:400 for WB, 1:100 for IF anti-Plexin-B2, intracellular domain, Abcam ab193355 (rabbit), 1:1000 for WB anti-Plexin-B3, R&D systems AF4958 (sheep), 1:400 for WB anti-Sema4B, Proteintech 16934-1-AP (rabbit), 1:400 for WB anti-Sema4C, LSBio C150056 (sheep), 1:400 for WB anti-YAP/TAZ, Santa Cruz sc101199 (mouse), 1:100 for IF.

Techniques: Dispersion, Staining, Comparison, Labeling, Fluorescence, Expressing, Migration

Journal: Communications Biology

Article Title: Plexin-B2 facilitates glioblastoma infiltration by modulating cell biomechanics

doi: 10.1038/s42003-021-01667-4

Figure Lengend Snippet: a Durotaxis stripe assay. SD2 GSCs expressing mCherry were cultured for 10 days on alternating stripes of soft (~25 kPa) vs. stiff (~30 kPa) PEG substrates. While control GSCs spread on both soft and stiff stripes, PB2-KO cells aggregated only on stiffer stripes. Enlarged images are shown on the right. Quantification measures mCherry fluorescence signals from soft vs. stiff stripes. n = 3 independent experiments per group. * P < 0.05, unpaired t test. b Still frames captured from time-lapse live imaging of GSCs, tracked with Hoechst nuclear dye (colored lines track individual nuclei). Quantifications reveal reduced migration distance over 90 min for PB2-KO GSCs as compared to controls (see Supplementary Videos). SD2 PB2-OE GSCs also had reduced speed of locomotion, but no significant change for SD3 was detected. n = 90 tracked nuclei per condition; one-way ANOVA with Dunnett’s multi-comparison test; * P < 0.05; ** P < 0.01. c Top, still frames from time-lapse live imaging of the indicated SD2 GSCs labeled with CellMask membrane dye. Bottom, three selected cells in each group shown in overlapping contour plots at 30 min-intervals over 90 min. Note the dynamic movement of control cells as compared to both PB2-KO and -OE conditions (see supplementary videos). d IF images show increased levels of phospho-myosin light chain 2 (pMLC2, arrows) in PB2-OE SD2 GSCs as compared to control cells. Right, quantifications show increased levels of IF intensity for pMLC2 per cell (corrected total fluorescence quantification; a.u., arbitrary unit). n > 30 cells for each condition. One-way ANOVA with Dunnett’s multi-comparison test; * P < 0.05. Differences for SD3 GSC did not reach statistical significance. e Live-cell imaging of GSCs stained with F-actin dye SPY-actin. Overexpression of PB2 in SD2 and SD3 GSCs leads to increased actin filament formation (arrowheads). Quantification indicates corrected total cell fluorescence; a.u., arbitrary unit. n > 30 cells for each condition. One-way ANOVA with Dunnett’s multi-comparison test; * P < 0.05. f Working model. Plexin-B2 controls actomyosin dynamics and interactions of invading GBM cells with neighboring cells and matrix substrates along migratory paths.

Article Snippet: Antibodies used for western blot analysis (WB) or immunofluorescence (IF) were as follows: anti-β-actin, Sigma A1978 (host species: mouse), dilution 1:10,000 for WB anti-EGFR, Cell Signaling Technology 4267 (rabbit), 1:1000 for WB anti-human nuclear antigen (HNA), Millipore MAB1281 (mouse), 1:250 for IF anti-integrin β1 (human-specific clone TS2/16), Santa Cruz sc-53711 (mouse), 1:300 for IF anti-laminin, Millipore MAB1905 (rat), 1:200 for IF anti-MBP, CST 78896 (rabbit), 1:100 for IF anti-MET, Cell Signaling Technology 8198 (rabbit), 1:1000 for WB anti-pMLC2 (Ser19), Cell Signaling Technology 3671 (rabbit), 1:100 for IF anti-PDGFRα, Cell Signaling Technology 3174 (rabbit), 1:1000 for WB anti-PECAM-1/CD31, BD Biosciences 553370 (rat), 1:300 for IF anti-Plexin-B1, R&D systems AF3749 (goat), 1:400 for WB anti-Plexin-B2, extracellular domain, R&D systems AF5329 (sheep), 1:400 for WB, 1:100 for IF anti-Plexin-B2, intracellular domain, Abcam ab193355 (rabbit), 1:1000 for WB anti-Plexin-B3, R&D systems AF4958 (sheep), 1:400 for WB anti-Sema4B, Proteintech 16934-1-AP (rabbit), 1:400 for WB anti-Sema4C, LSBio C150056 (sheep), 1:400 for WB anti-YAP/TAZ, Santa Cruz sc101199 (mouse), 1:100 for IF.

Techniques: Stripping Membranes, Expressing, Cell Culture, Fluorescence, Imaging, Migration, Comparison, Labeling, Membrane, Live Cell Imaging, Staining, Over Expression

Journal: Communications Biology

Article Title: Plexin-B2 facilitates glioblastoma infiltration by modulating cell biomechanics

doi: 10.1038/s42003-021-01667-4

Figure Lengend Snippet: a Genes that are correlated with PLXNB2 expression levels in GBM patients (Spearman correlation FDR < 10%, TCGA database) are enriched for pathways concerning cell adhesion, cell-substrate junctions, and actomyosin. Left, top 10 significant functional terms (MSigDB) are shown. Right, diagram of connectivity of coregulated genes with PLXNB2 in human GBMs, filtered for genes involved in cell adhesion, motility/invasion, and EMT. b Examples of RNA-seq read tracks for PLXNB2 mRNA in three GSC clonal lines of wild-type and PB2-KO genotypes from SD4 GSCs. Frameshift mutations in the PB2-KO lines lead to the reduction of PLXNB2 mRNA levels by nonsense-mediated decay (NMD). c Pathway enrichment analyses of up- and downregulated DEGs in response to Plexin-B2 KO (union of all DEGs from four GSC lines; FDR < 20%). Top ten significantly enriched terms (MSigDB) are shown. Dashed yellow lines denote −log10 (adjusted P value) = 0.05. d GSEA of expression changes in PB2-KO vs. control GSCs shows enrichment of Matrisome gene set (Naba et al. ) for all four GSC lines. The sinusoidal shapes of enrichment scores (green line) indicate enrichment of both up- and downregulated genes.

Article Snippet: Antibodies used for western blot analysis (WB) or immunofluorescence (IF) were as follows: anti-β-actin, Sigma A1978 (host species: mouse), dilution 1:10,000 for WB anti-EGFR, Cell Signaling Technology 4267 (rabbit), 1:1000 for WB anti-human nuclear antigen (HNA), Millipore MAB1281 (mouse), 1:250 for IF anti-integrin β1 (human-specific clone TS2/16), Santa Cruz sc-53711 (mouse), 1:300 for IF anti-laminin, Millipore MAB1905 (rat), 1:200 for IF anti-MBP, CST 78896 (rabbit), 1:100 for IF anti-MET, Cell Signaling Technology 8198 (rabbit), 1:1000 for WB anti-pMLC2 (Ser19), Cell Signaling Technology 3671 (rabbit), 1:100 for IF anti-PDGFRα, Cell Signaling Technology 3174 (rabbit), 1:1000 for WB anti-PECAM-1/CD31, BD Biosciences 553370 (rat), 1:300 for IF anti-Plexin-B1, R&D systems AF3749 (goat), 1:400 for WB anti-Plexin-B2, extracellular domain, R&D systems AF5329 (sheep), 1:400 for WB, 1:100 for IF anti-Plexin-B2, intracellular domain, Abcam ab193355 (rabbit), 1:1000 for WB anti-Plexin-B3, R&D systems AF4958 (sheep), 1:400 for WB anti-Sema4B, Proteintech 16934-1-AP (rabbit), 1:400 for WB anti-Sema4C, LSBio C150056 (sheep), 1:400 for WB anti-YAP/TAZ, Santa Cruz sc101199 (mouse), 1:100 for IF.

Techniques: Expressing, Functional Assay, RNA Sequencing Assay

Journal: Communications Biology

Article Title: Plexin-B2 facilitates glioblastoma infiltration by modulating cell biomechanics

doi: 10.1038/s42003-021-01667-4

Figure Lengend Snippet: a Cartoon of Plexin-B2 domain structure. Cell membrane shown as a gray bar; dashed line indicates proteolytic cleavage into α and β chains. Sema, Sema domain; PSI, plexin-semaphorin-integrin domain; IPT, Ig-like fold shared by plexins and transcription factors; RBD, Rho-binding domain; GAP, GTPase activating protein; VTDL, PDZ-domain binding site formed by four C-terminal amino acids for docking of PDZ-Rho-GEF or LARG proteins. b Diagrams of lentiviral vectors encoding cDNA of wild-type or signaling mutants of Plexin-B2. PB2* is CRISPR-resistant cDNA, due to synonymous mutations (X) at sgRNA target site. dEcto lacks extracellular domain, mGAP has mutated GTPase activation domain, mRBD has mutated Rho-binding domain, and dVTDL has deleted PDZ-binding motif. c Cell aggregation assay in hanging drops used for Plexin-B2 (PB2) rescue experiments. Photos and enlargements show aggregates formed from GSCs with the indicated Plexin-B2 mutations in hanging drops after 96 h. Quantifications of aggregate numbers and sizes in hanging drops at 24 or 96 h are shown below. The mGAP and dEcto Plexin-B2 mutants failed to rescue the KO phenotype (marked as blue). n = 5–11 drops per group; one-way ANOVA with Dunnett’s multiple comparison test of each group against SD2-Ctrl; * P < 0.05, ***0.001. d Left, representative micrographs of cell dispersion assay with GSCs expressing mutant Plexin-B2 rescue constructs. Dispersed cells from aggregates were visualized at 4 h with DAPI nuclear stain. Bottom, quantifications show the number of nuclei detached from spheres normalized to mean in the control condition. The PB2 mGAP mutant failed to rescue the KO phenotype (marked as blue). Box plots and whiskers indicate quartiles, center lines indicate median. n = 18–34 spheres per group; one-way ANOVA with Dunnett’s multiple comparison test of each group against control; * P < 0.05, *** P < 0.001. e Model depicting Plexin-B2 signaling through Ras-GAP domain to affect cell biomechanics during GBM invasion.

Article Snippet: Antibodies used for western blot analysis (WB) or immunofluorescence (IF) were as follows: anti-β-actin, Sigma A1978 (host species: mouse), dilution 1:10,000 for WB anti-EGFR, Cell Signaling Technology 4267 (rabbit), 1:1000 for WB anti-human nuclear antigen (HNA), Millipore MAB1281 (mouse), 1:250 for IF anti-integrin β1 (human-specific clone TS2/16), Santa Cruz sc-53711 (mouse), 1:300 for IF anti-laminin, Millipore MAB1905 (rat), 1:200 for IF anti-MBP, CST 78896 (rabbit), 1:100 for IF anti-MET, Cell Signaling Technology 8198 (rabbit), 1:1000 for WB anti-pMLC2 (Ser19), Cell Signaling Technology 3671 (rabbit), 1:100 for IF anti-PDGFRα, Cell Signaling Technology 3174 (rabbit), 1:1000 for WB anti-PECAM-1/CD31, BD Biosciences 553370 (rat), 1:300 for IF anti-Plexin-B1, R&D systems AF3749 (goat), 1:400 for WB anti-Plexin-B2, extracellular domain, R&D systems AF5329 (sheep), 1:400 for WB, 1:100 for IF anti-Plexin-B2, intracellular domain, Abcam ab193355 (rabbit), 1:1000 for WB anti-Plexin-B3, R&D systems AF4958 (sheep), 1:400 for WB anti-Sema4B, Proteintech 16934-1-AP (rabbit), 1:400 for WB anti-Sema4C, LSBio C150056 (sheep), 1:400 for WB anti-YAP/TAZ, Santa Cruz sc101199 (mouse), 1:100 for IF.

Techniques: Membrane, Binding Assay, CRISPR, Activation Assay, Comparison, Dispersion, Expressing, Mutagenesis, Construct, Staining

Journal: G3: Genes|Genomes|Genetics

Article Title: Global Transcriptome Changes That Accompany Alterations in Serotonin Levels in Caenorhabditis elegans

doi: 10.1534/g3.120.401088

Figure Lengend Snippet: 5-HT treatment induced similar changes as seen in stressed animals. (A, B) Scatterplot showing the relationship between significantly enriched genes from 5-HT-treated animals and genes from a SPELL transcription dataset from the two enriched categories. Line represents linear regression, Pearson’s r value shown. p-value is corrected for multiple tests. (A) the relationship between significantly enriched genes from 5-HT-treated animals and animals in SPELL dataset belonging to the ‘Immune’ category. (B) the relationship between significantly enriched genes from 5-HT-treated animals and animals in the SPELL dataset belonging to the ‘Defense’ category. (C) Table showing the list of datasets and publications that showed a significant correlation with the 5-HT RNA-seq data. The left column indicates the Wombase ID and the right column the SPELL ID. (D) Phospho- eIF2α (p (Ser51)-eIF2α) levels in animals with elevated 5-HT. Top Panel shows representative Western Blot using anti-phospho (Ser51)-eIF2-α antibody. Lower Panel show Bar chart of the relative expression p(Ser51)-eIF2α normalized to the untreated control. Tubulin was used as a loading control. Bars in D represent mean, error bars correspond to standard error of the mean. Significance was determined using a student’s two-tailed t -test. P < 0.05.

Article Snippet: Rabbit anti-phospho (Ser51)-eIF2-α antibody (catalog no. 9721S, Cell Signaling Technology) was used to detect the level of phosphorylated eIF2-α.

Techniques: RNA Sequencing, Western Blot, Expressing, Control, Two Tailed Test

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: ( A ) Representative micrographs of negative, low, moderate, and high CD109 IHC staining of clinical glioma samples. Scale bar: 50 μm. ( B ) Association of CD109 expression with tumor grade ( n = 346). P < 0.007, χ 2 test. See also . ( C ) Kaplan-Meier survival analysis based on CD109 expression. The median value was used as cutoff; CD109 hi ( n = 118), red line, and CD109 lo ( n = 122), black line. P = 0.024, log-rank test. ( D and E ) Association of CD109 expression with p-STAT3 ( n = 78; P = 0.002) ( D ) and Ki-67 ( n = 173; P = 0.041) ( E ) in glioblastomas, χ 2 test. See also . ( F ) CD109 mRNA expression between glioblastoma ( n = 489) and nontumor specimens ( n = 10) in the TCGA glioblastoma (TCGA GBM) data set. P < 0.001, Tukey’s post hoc test. ( G ) Heatmap shows CD109 mRNA expression between different glioblastoma subtypes across several glioblastoma data sets: mesenchymal (MES), classical (CL), and proneural (PN). ( H ) Heatmap shows mRNA expression levels of CD109 , IL6ST , STAT3 , and IL-6 between glioblastoma cell lines across different subtypes in the HGCC data set.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Immunohistochemistry, Expressing

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: ( A ) Heatmap shows the classification of patient-derived GSCs and the H2 cell line based on their dominant transcriptional subtype: mesenchymal-like (MES), classical-like (CL), and proneural-like (PN). ( B and C ) Western blot analyses of CD109, SOX2, and OLIG2 expression in GSCs and differentiated glioma cells cultured in FBS-containing medium at the indicated time points. ( D ) Western blot analysis of CD109 expression and p-STAT3 levels in GSCs of different glioblastoma subtypes. ( E and F ) Western blot analyses of SOX2 and OLIG2 expression in CD109-silenced GSCs and nontargeted controls at the indicated time points. ( G and H ) Real-time quantitative PCR (qRT-PCR) analysis of GSC marker mRNA levels in CD109-silenced and nontargeted control GSCs at day 11. ( I ) Cell proliferation of CD109-silenced and nontargeted control GSCs at indicated time points. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; **** P < 0.0001, 2-way ANOVA with Tukey’s multiple comparisons test. ( J ) Representative micrographs of colony formation in methylcellulose after CD109 silencing. Scale bar: 200 μm. ( K and L ) Quantification of colonies in methylcellulose. Data are presented as mean ± SD. **** P < 0.0001, 1-way ANOVA with Dunnett’s multiple comparisons test. ( M ) Representative micrographs of GSC growth in 3D fibrin matrix after CD109 silencing. Scale bar: 200 μm. Inset: original magnification, ×10. See also . ( N ) Cell viability in 3D fibrin matrix at day 15. Data are presented as mean ± SD. **** P < 0.0001, unpaired 2-tailed t test. See also . ( O ) Western blot analysis of p-STAT3 levels after CD109 silencing. Data are from n = 2 ( B and C ) and n = 3 ( D – O ) independent experiments. Representative Western blots are shown where β-tubulin served as a loading control.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Derivative Assay, Western Blot, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Marker

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: ( A ) qRT-PCR analysis of IL6R and IL6ST mRNA levels in GSCs. Data are presented as mean ± SD. ( B and C ) Western blot analyses of p-STAT3 levels in CD109-silenced and nontargeted control GSCs after stimulation with IL-6 cytokine (50 ng/mL) for 15 and 30 minutes. ( D ) qRT-PCR analysis of IL6ST mRNA levels in CD109-silenced and nontargeted control GSCs. Data are presented as mean ± SEM. * P < 0.05; **** P < 0.0001, unpaired 2-tailed t test. ( E ) Western blot analysis of GP130 expression after CD109 silencing in GSCs of different glioblastoma subtypes. ( F and G ) Western blot analyses of CD109 expression and p-STAT3 levels in GP130-silenced and nontargeted control GSCs. ( H ) Co-IP analysis of CD109. GSC whole-cell extracts were subjected to immunoprecipitation with an anti-GP130 antibody or appropriate IgG control followed by Western blotting with an anti-CD109 antibody. Inputs are indicated. ( I and J ) Representative micrographs of PLA in GSCs using anti-CD109 and anti-GP130 antibodies. Appropriate IgG controls served as negative controls. Red indicates specific interaction signal. Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. ( K and L ) Quantification of PLA signal per cell (DAPI). Data are presented as mean ± SD. ** P < 0.01; **** P < 0.0001, 1-way ANOVA with Tukey’s multiple comparisons test. Data are from n = 3 ( A – G ) and n = 2 ( H – L ) independent experiments. Representative Western blots are shown, where β-tubulin served as a loading control.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: ( A and B ) qRT-PCR analysis of MAP2 , GALC , and GFAP mRNA levels in CD109-silenced and nontargeted GSCs at day 11. Data are presented as mean ± SEM. ** P < 0.01; **** P < 0.0001, 2-way ANOVA with Dunnett’s multiple comparisons test. ( C ) Heatmap shows increased expression of AC-like genes after CD109 silencing at day 11. Data are presented as fold change relative to control. ( D and E ) qRT-PCR analysis of CL subtype genes in CD109-silenced and nontargeted GSCs at d11. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, unpaired 2-tailed t test. ( F ) Cell viability of CD109-silenced and nontargeted GSCs at the indicated time points. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01, 1-way ANOVA with Kruskal-Wallis post hoc test. ( G and H ) Immunofluorescence staining of cleaved caspase-3 (green) and F-actin (red) in CD109-silenced and nontargeting control GSCs in 3D fibrin matrix at day 15. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. ( I ) Box-and-whisker plot shows cell viability of CD109-silenced and nontargeting control GSCs after treatment with 250 μM of TMZ for 4 days. Data were normalized to the corresponding vehicle control. **** P < 0.0001, nonparametric Mann-Whitney U test (BT12) and unpaired 2-tailed t test (BT13 and S24). ( J ) Box-and-whisker plot shows cell viability after treatment with Stattic or combination of Stattic and TMZ. Data were normalized to the corresponding vehicle control. * P < 0.05; ** P < 0.01, nonparametric Mann-Whitney U test. Box plots represent the first quartile, median, and third quartile, with whiskers indicating minimum and maximum values. Data are from n = 3 ( A , B , and D – J ) independent experiments.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Whisker Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: Representative micrographs of coronal sections of tumor-bearing animal brains after implantation of nontargeted control or CD109-silenced BT12 ( A ), BT13 ( B ), BT18 ( C ), or ZH305 ( D ) GSCs. Frozen sections were immunostained with anti-human vimentin (green). Nuclei were counterstained with DAPI (white). Scale bar: 2 mm. Quantification of the BT12 ( E ) and BT13 ( F ) tumor volume, number of BT12 ( G ) and BT13 ( H ) satellite tumors, and number of single invasive BT12 ( I ) and BT13 ( J ) cells of control and CD109-silenced BT12 ( n = 10) and BT13 ( n = 10) xenografts. Quantification of the BT18 ( K ) and ZH305 ( L ) tumor area and number of single invasive BT18 ( M ) and ZH305 ( N ) cells of control and CD109-silenced BT18 ( n = 7) and ZH305 ( n = 6) xenografts. Quantification of the number of Ki-67 + BT12 ( O ) and BT13 ( P ) tumor cells and number or OLIG2 + BT12 ( Q ) and BT13 ( R ) tumor cells of control and CD109-silenced BT12 ( n = 10) and BT13 ( n = 10) xenografts. Western blot analysis of expression of indicated proteins from the whole brain section extracts of BT12 ( S ) and BT13 ( T ) control and CD109-silenced BT12 ( n = 3) and BT13 ( n = 3) xenografts. ( U ) Representative micrographs of BT12 xenografts immunostained with anti–PDGFR-α (white), anti-human vimentin (green), and podocalyxin (red). Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. ( V ) Representative micrographs of BT12 xenografts immunostained with anti-NG2 (red, arrows) and anti-human nuclear marker (NUMA, green). Nuclei were counterstained with DAPI (blue). Scale bars: 1 mm or 25 μm as indicated. ( W ) Quantification of the number of PDGFR-α + cells in control and CD109-silenced BT12 ( n = 10) xenografts. ( X ) Quantification of the number of NG2 + cells in control and CD109-silenced BT12 ( n = 10) xenografts. P values were calculated using unpaired 2-tailed t test.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Western Blot, Expressing, Marker

Journal: JCI Insight

Article Title: CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity

doi: 10.1172/jci.insight.141486

Figure Lengend Snippet: ( A ) Representative micrographs of BT12 xenografts immunostained with anti–collagen IV (green) and anti-podocalyxin (red). Nuclei were counterstained with DAPI (blue). Scale bar: 40 μm. Arrows indicate empty basement membrane sleeves devoid of endothelium. ( B ) Quantification of collagen IV + /PODXL – empty sleeves in control and CD109-silenced BT12 xenografts ( n = 10). ( C ) Representative micrographs of BT12 xenografts immunostained with anti-human vimentin (green) and anti-mouse CD31 (red). Nuclei were counterstained with DAPI (blue). Scale bars: 2 mm or 40 μm as indicated. Quantification of the average tumor blood vessel size ( D ) and density ( E ) in control and CD109-silenced BT12 xenografts ( n = 10). ( F ) Quantification of the secreted ANG1 and ANG2 levels from the cell culture supernatants of CD109-silenced and nontargeted control BT12 GSCs ( n = 2). ( G ) Expression of ANGPT1 and ANGPT2 mRNA levels determined by total RNA-Seq in CD109-silenced and nontargeted BT12 GSCs at the indicated time points. ( H ) Representative micrographs of BT12 xenografts immunostained with anti-human NUMA (green) and anti-NG2 (red). Nuclei were counterstained with DAPI (blue). Scale bars: 2 mm or 40 μm as indicated. ( I ) Representative micrographs of BT12 xenografts immunostained with anti–α-SMA (green) and anti-CD31 (red). Nuclei were counterstained with DAPI (blue). Scale bar: 40 μm. ( J ) Quantification of the α-SMA + tumor pericytes in control and CD109-silenced BT12 xenografts ( n = 10). ( K ) Representative micrographs of BT12 xenografts immunostained with anti-NG2 (green) and anti-CD31 (red). Nuclei were counterstained with DAPI (blue). Scale bar: 40 μm. ( L ) Quantification of the pericyte coverage in control and CD109-silenced BT12 xenografts ( n = 10). P values were calculated by using the unpaired 2-tailed t test except for F and G , where a t test with Mann-Whitney post hoc test was used.

Article Snippet: Immunohistochemistry: polyclonal sheep CD109 antibody and polyclonal goat OLIG2 antibodies (AF-4385 and AF-2418, R&D Systems, Bio-Techne), monoclonal mouse Ki-67 (M7240, Dako).

Techniques: Membrane, Cell Culture, Expressing, RNA Sequencing Assay, MANN-WHITNEY