Journal: bioRxiv

Article Title: Therapeutic efficacy of an immune stimulatory gene therapy strategy in a mouse model of high grade brainstem glioma

doi: 10.1101/846097

Figure Lengend Snippet: (A) Schematic representation of SB transposase and transposon plasmids used to develop gliomas in the brainstem. Black arrows indicate position of inverted repeat and direct repeat (IR/DR) sequences which flank specific DNA sequences (Luciferin, NRAS GV12, shP53-GFP, ACVR1 G328V-IRES-Katushka). (B) Schematic representation of brainstem glioma model. (i) One day old pups were injected in the fourth ventricle (3 mm posterior to the λ-suture and 3 mm deep) with plasmid cocktail. (ii) Bioluminescence imaging at 1-day post injection (dpi) confirming the efficiency of the in vivo transfection. (iii) Bioluminescence imaging obtained when mice displayed signs of neurological deficits confirms the presence of a large tumor. (iv) Fluorescence image of GFP + brainstem tumor. (C) Kaplan-Meier survival curve for genetically engineered mice bearing mACVR1 (n= 11) tumor; MS: median survival. (D) Hematoxylin and eosin stained paraffin embedded SB brainstem tumor sections (wt-ACVR1 and mACVR1). Scale bar in the upper panel images is 200 µm. Scale bar in the bottom panel images is 20 µm. (E-J) Immunohistochemistry (IHC) staining for: (E) Olig2, an oligodendrocyte marker (scale bar in the upper panel image is 200 µm, scale bar for the bottom panel images is 50 µm) (F) GFAP, an astrocyte marker (scale bar in the upper panel image is 200 µm, scale bar for the bottom panel images is 20 µm) (G) Nestin, a neural stem cell marker (scale bar in the upper panel image is 200 µm, scale bar for the bottom panel images is 20 µm) (H) Sox2, a transcription factor that plays an important role in the maintenance of neural stem cells (scale bar in the upper panel image is 200 µm, scale bar for the bottom panel images is 50 µm) (I) Ki67, a marker of proliferating cells (scale bar in the upper panel image is 200 µm, scale bar for the bottom panel images is 20 µm) and (J) phosphorylated ERK-1/2 protein (pERK1/2) (scale bar in the upper panel image is 200 µm, scale bar for the bottom panel images is 50 µm).

Article Snippet: The pCMV5-ALK2-WT plasmid was a generous gift from Jeff Wrana (Addgene plasmid #11741).

Techniques: Injection, Plasmid Preparation, Imaging, In Vivo, Transfection, Fluorescence, Staining, Immunohistochemistry, Marker

Journal: bioRxiv

Article Title: Therapeutic efficacy of an immune stimulatory gene therapy strategy in a mouse model of high grade brainstem glioma

doi: 10.1101/846097

Figure Lengend Snippet: (A) Diagram representing the TGF-ß/Smad signaling pathway. When ACVR1 is mutated this pathway is constitutively active regardless of the presence or absence of BMP ligand. LDN-214117 inhibits ACVR1 kinase activity. (B) Immunofluorescence (IF) staining for phospho-Smad1/5 in paraffin embedded SB brainstem tumor sections (wt-ACVR1 and mACVR1). Scale bar in the upper panel image is 100 µm, scale bar for the bottom panel images is 50 µm. White arrows indicate positive expression. (C) Immunohistochemistry (IHC) staining for Id1 protein in paraffin embedded SB brainstem tumor sections (wt-ACVR1 and mACVR1). Scale bar in the upper panel image is 100 µm, scale bar for the bottom panel images is 50 µm. (D) WB assay was performed on three different clones of wt-ACVR1 and mACVR1 NS to assess the protein level of phopho-Smad1/5, Smad1, and Id1, a downstream Smad1/5 regulated gene. ß-actin: loading control. C1, C2, C3 represent the clones of NS used. (E) Western blot assay was performed on mACVR1 NS treated with LDN-214117 (0.03-1uM) and blotted for Smad1/5, Smad1, and Id2; ß-actin: loading control.

Article Snippet: The pCMV5-ALK2-WT plasmid was a generous gift from Jeff Wrana (Addgene plasmid #11741).

Techniques: Activity Assay, Immunofluorescence, Staining, Expressing, Immunohistochemistry, Clone Assay, Western Blot

Journal: bioRxiv

Article Title: Therapeutic efficacy of an immune stimulatory gene therapy strategy in a mouse model of high grade brainstem glioma

doi: 10.1101/846097

Figure Lengend Snippet: (A) Differential gene expression in mACVR1 tumors analyzed by RNA-seq. Volcano plot comparing differentially expressed genes in mACVR1 versus wt-ACVR1 mouse NS. The log 10 (FDR corrected p-values); q-values were plotted against the log 2 (Fold Change: FC) in gene expression. Genes upregulated by ≥ 1.3 fold and with a FDR corrected p-value < 0.05 are depicted as red dots; genes that were downregulated by ≥1.3 fold and with a FDR corrected p-value < 0.05 are depicted as green dots. (B) Gene Ontology (GO) enrichment analysis performed using iPathwayGuide for up- and down-regulated genes. The bar graph shows the GO biological process, selected for relevance to phenotype, that are differentially expressed between mACVR1 versus wt-ACVR1 NS. The –log10 q values of GO terms were plotted; FDR corrected P value = q value. (C) Differentially expressed pathway genes associated with mACVR1 in mACVR1 versus wt-ACVR1 mouse NS. Plot of the log (FC) of genes with an FDR corrected p-value < 0.05. (D) Pathway enrichment maps of DE genes in mACVR1 versus wt-ACVR1 NS. Clusters of nodes depicted in red illustrate differentially upregulated pathways resulting from GSEA (P < 0.05; FDR < 0.5). The yellow highlighted nodes indicate up-regulated GO terms containing ACVR1. (E) GSEA enrichment plot of regulation of BMP signaling pathway genes identified by RNA-Seq analysis in mACVR1 versus wt-ACVR1 mouse NS. (F) GSEA enrichment plot of regulation of cell differentiation genes identified by RNA-Seq analysis in mACVR1 versus wt-ACVR1 mouse NS.

Article Snippet: The pCMV5-ALK2-WT plasmid was a generous gift from Jeff Wrana (Addgene plasmid #11741).

Techniques: Expressing, RNA Sequencing Assay, Cell Differentiation

Journal: bioRxiv

Article Title: Therapeutic efficacy of an immune stimulatory gene therapy strategy in a mouse model of high grade brainstem glioma

doi: 10.1101/846097

Figure Lengend Snippet: (A) Levels of CD133, CD44, and Aldh1 in the (tumor microenvironment) TME of mice bearing wt-ACVR1 or mACVR1 was assessed when animals displayed signs of tumor burden. Representative histograms display each marker’s expression (green = wt-ACVR1, red = mACVR1). MFI = mean fluorescence intensity; * p < 0.05; *** p < 0.001; unpaired t test. Bars represent mean ± standard error of the mean (SEM) (n = 3 biological replicates). ( B-C ) In vivo tumor initiation capacity. Kaplan-Meier survival curves for mice intracranially implanted with different numbers of wt-ACVR1 (B) or mACVR1 NS (C) as indicated in the plot. MS: median survival.

Article Snippet: The pCMV5-ALK2-WT plasmid was a generous gift from Jeff Wrana (Addgene plasmid #11741).

Techniques: Expressing, Fluorescence, In Vivo

Journal: bioRxiv

Article Title: Therapeutic efficacy of an immune stimulatory gene therapy strategy in a mouse model of high grade brainstem glioma

doi: 10.1101/846097

Figure Lengend Snippet: (A) wt-ACVR1 or mACVR1 NS were treated with TK (500 MOI) and/ or 3 Gray (Gy) ionizing radiation (IR), followed by GCV (25 µM). Levels of calreticulin or HMGB1 on tumor NS were assessed via flow cytometry analysis 24hrs post treatment. MFI = mean fluorescence intensity; * p < 0.05; **** p < 0.0001; two-way ANOVA, followed by Turkey’s multiple comparisons. Levels of ATP released into the media were assessed using a colorimetric assay. (B) Experimental design showing mice bearing mACVR1 brainstem gliomas treated with saline or TK/FLT3L gene therapy on day 5 post tumor implantation, followed by intraperitoneal administration of GCV (25 mg/kg) on day 6-12. Radiation was administered at a dose of 2Gy/Day for 5d for two weeks 5 days’ post tumor implantation; mice were monitored for survival. (C) Kaplan Meier survival analysis for mice bearing mACVR1 tumors treated with saline (n = 5), IR (n = 6), TK+ GCV (n=5), or IR+TK+GCV (n=5). Data were analyzed using log-rank (Mantel-Cox) test; ** p < 0.005.

Article Snippet: The pCMV5-ALK2-WT plasmid was a generous gift from Jeff Wrana (Addgene plasmid #11741).

Techniques: Flow Cytometry, Fluorescence, Colorimetric Assay, Tumor Implantation

Journal: REPRODUCTION

Article Title: Postnatal uterine development in Inverdale ewe lambs

doi: 10.1530/rep-07-0323

Figure Lengend Snippet: Figure 3 Immunohistochemical localization of androgen receptor (AR) and activin receptors (ActRIA, IB and II) in the uteri of CC and II ewes. GE, glandular epithelium; LE, luminal epithelium; S, stroma. The scale bar indicates 100 mm at low magnification and 25 mm at high magnification.

Article Snippet: Rabbit polyclonal antibody to androgen receptor (sc-816, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and mouse monoclonal Reproduction (2008) 135 357–365 antibodies to ActRIA (MAB637; R&D Systems Inc.), ActRIB (MAB222; R&D Systems), and ActRII (MAB3391; R&D Systems) were used for immunohistochemistry (Carpenter et al. 2003a, Hayashi et al. 2003, Juengel et al. 2006).

Techniques: Immunohistochemical staining

Journal: Biology of reproduction

Article Title: The activin-follistatin system in the neonatal ovine uterus.

doi: 10.1095/biolreprod.103.016287

Figure Lengend Snippet: FIG. 2. Expression of ActRs in the neonatal ovine uterus. In situ hybridization and immunohistochemical analysis of ActRIA (A) and ActRII (B) and immunohistochemical analysis of ActRIB (C) in the uterus. Except for ActRIB, representative photomicrographs of in situ hybridization results are presented in bright-field and dark-field illumination (left). Melanocytes (Mel) in the endometrium appear white in the dark-field images and black in bright-field images, but they do not express subunit mRNA. Representative photomicrographs of immunohistochemical results are presented for the upper and lower portions of the uterine wall (right). As a negative control, mouse IgG (mIgG) was substituted for the primary antibody. M, Myometrium; S, stroma. Bars 5 50 mm.

Article Snippet: Mouse anti-human monoclonal antibody to follistatin (catalog no. MAB669), ActRIA (catalog no. MAB637), ActRIB (catalog no. MAB222), and ActRIIA/B (catalog no. MAB3391) were from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Expressing, In Situ Hybridization, Immunohistochemical staining, Negative Control

Journal: Biology

Article Title: BMP-2-Driven Osteogenesis: A Comparative Analysis of Porcine BMSCs and ASCs and the Role of TGF-β and FGF Signaling.

doi: 10.3390/biology14060610

Figure Lengend Snippet: Figure 6. Receptor expressions of ALK2, ALK4, ALK6, ALK5, ALK7, BMPR-II in pASC. Grouped representation of the respective receptor expression in the course of osteogenic differentiation (OM +/−BMP-2). From day 19, there is a significant induction of ALK 2, ALK 6, and ALK 5 with the addition of BMP-2. BMPR-II expression in the OM group decreased in OM and tended to stay increased under BMP-2 supplementation from day 19, but was not considered significant (* p ≤0.05, ** p ≤0.01; n = 6, BMP-2 450 ng/mL).

Article Snippet: The respective conjugated antibodies were used for the expressions of ALK3 (Cat. No.: AF436), ALK 5 (Cat. No.: FAB5871), ALK6 (Cat. No.: FAB5051A), TGF-β2-RII (Cat. No.: FAB532P), ALK7 (Cat. No.: FAB77491A), ALK2 (Cat. No.: AF637), ALK4 (Cat. No.: MAB2221), and BMPR-II (Cat. No.: AF811) (by R&D Systems, Minneapolis, MN, USA), and the pASCs and pBMSCs were compared for their expressions of the specific surface antigens CD45 (Cat. No.: MCA1568GA, BioRad, Hercules, CA, USA), HLA-DR (human leukocyte antigen–antigen D-related surface molecule) (Cat. No.: MCA2314F, Bio-Rad, Hercules, CA, USA), CD29 (Cat. No.: 561,496, BD Pharmingen, Franklin Lakes, NJ, USA), CD79alpha (Bio-Rad, Cat. No.: MCA2538GA), CD14 (Cat. No.: MCA1568GA, Bio-Rad, Hercules, CA, USA), CD31 (Cat. No.: AF3387, R&D Systems, Minneapolis, MN, USA), CD105 (Cat. No.: NB110-58718APC, Novus Biologicals, Minneapolis, MN, USA), CD26 (, Cat. No.: NB600-552APC, Novus Biologicals, Minneapolis, MN, USA), CD73 (, Cat. No.: AF4488, R&D Systems, Minneapolis, MN, USA), CD90 (Cat. No.: 559,869, BD Pharmingen, Franklin Lakes, NJ, USA), CD34 (Cat. No.: 81289, abcam, Cambridge, UK), and CD44 (Cat. No.: 5531, BD Pharmingen, Franklin Lakes, NJ, USA).

Techniques: Expressing