Journal: The Journal of Physiology

Article Title: Forkhead BoxO transcription factors restrain exercise-induced angiogenesis

doi: 10.1113/jphysiol.2014.275867

Figure Lengend Snippet: A, FVB/n mice (n = 4) exercised for 1 day or remained sedentary (S). Muscles were extracted immediately (Ex), or 2 h after training (Ex+2 h). B, FVB/n FoxOL/L mice (n = 6) trained for 1, 7, 10 or 14 days (D1, D7, D10, D14) or remained sedentary (S), and all muscles were extracted 2 h after the final training session. In both, THBS1 protein was assessed relative to β-actin. For densitometric analysis of THBS1, intensities of the three bands evident at ∼ 175 kDa were summed together. *P < 0.05 vs. sedentary; ##P < 0.01 vs. Ex (Tukey post hoc analysis).

Article Snippet: RNA was reverse transcribed using standard protocols. qPCR cDNA samples were analysed by qPCR with an ABI 7500 Fast PCR System (Invitrogen Canada, Burlington, ON, Canada) using qPCR mastermix (P/N 11743; Invitrogen Canada) and Taqman probe/primer sets (Invitrogen Canada) for FoxO1 (Mm00490672_m1), FoxO3a (Mm01185722_m1), THBS1 (Mm01335418_m1) and HPRT1 (Mm00446968_m1).

Techniques: Muscles

Journal: The Journal of Physiology

Article Title: Forkhead BoxO transcription factors restrain exercise-induced angiogenesis

doi: 10.1113/jphysiol.2014.275867

Figure Lengend Snippet: A, FoxO1 and FoxO3a protein levels were analysed in endothelial cells isolated from epididymal fat of FoxOL/L and FoxOΔ mice (n = 3). Representative blots are shown, with the respective densitometric values (relative to β-actin) listed under each blot. B, muscles were extracted from FoxOL/L and FoxOΔ sedentary (S) mice, or 2 h after an acute exercise bout (D1). FoxO1 and FoxO3a protein levels were assessed by Western blot to determine the extent of deletion from whole gastrocnemius extracts, and quantified relative to tubulin. Significant exercise and genotype effects were observed (two-way ANOVA) for both FoxO1 (P = 0.0008 and P = 0.006, respectively) and FoxO3a (P = 0.01 and P = 0.009, respectively). C, FoxOL/L and FoxOΔ mice (n = 6) trained for 1, 7 or 14 days, or remained sedentary (S) and muscles were extracted 2 h after training. THBS1 mRNA levels were measured by qPCR, normalizing to the housekeeping gene HPRT. Significant training (P < 0.0001) and genotype (P < 0.01) effects were observed (two-way ANOVA). D, THBS1 protein was assessed by Western blot of whole gastrocnemius extracts and quantified relative to tubulin. Significant exercise (P < 0.05) and genotype (P < 0.05) effects were observed (two-way ANOVA). *P < 0.05; ***P < 0.001 vs. sedentary FoxOL/L; #P < 0.05; ##P < 0.01; ###P < 0.001 vs. FoxOΔ day 1 (Bonferroni post hoc analysis).

Article Snippet: RNA was reverse transcribed using standard protocols. qPCR cDNA samples were analysed by qPCR with an ABI 7500 Fast PCR System (Invitrogen Canada, Burlington, ON, Canada) using qPCR mastermix (P/N 11743; Invitrogen Canada) and Taqman probe/primer sets (Invitrogen Canada) for FoxO1 (Mm00490672_m1), FoxO3a (Mm01185722_m1), THBS1 (Mm01335418_m1) and HPRT1 (Mm00446968_m1).

Techniques: Isolation, Muscles, Western Blot

Journal: Frontiers in Oncology

Article Title: TP53 Status is Associated with Thrombospondin1 Expression In vitro

doi: 10.3389/fonc.2013.00269

Figure Lengend Snippet: Relationship between THBS1 differential gene expression, promoter methylation, mRNA expression, and TP53 mutations in ovarian cancer cell lines .

Article Snippet: Primers and probes for THBS1 (Hs00170236_m1) and GAPDH (human 402869) were obtained from Applied Biosystems (Foster City, CA, USA).

Techniques: Gene Expression, Methylation, Expressing, Mutagenesis, Over Expression

Journal: Frontiers in Oncology

Article Title: TP53 Status is Associated with Thrombospondin1 Expression In vitro

doi: 10.3389/fonc.2013.00269

Figure Lengend Snippet: THBS1 gene expression, THBS1 promoter methylation, tumor growth properties, and chemotherapy-induced growth inhibition . Pyrosequencing shows low levels of methylation in the cell lines (median = 8.6%; range = 3.5 to 88.8%). (A) Cells with low levels of promoter methylation exhibited higher THBS1 gene expression, while those with high levels of promoter methylation had low THBS1 gene expression. There was no association between THBS1 gene expression with anchorage-independent growth; (B) population doubling time (C) invasive capacity; (D) or cisplatin; (E) and paclitaxel; (F) IC50 values.

Article Snippet: Primers and probes for THBS1 (Hs00170236_m1) and GAPDH (human 402869) were obtained from Applied Biosystems (Foster City, CA, USA).

Techniques: Gene Expression, Methylation, Inhibition

Journal: Frontiers in Oncology

Article Title: TP53 Status is Associated with Thrombospondin1 Expression In vitro

doi: 10.3389/fonc.2013.00269

Figure Lengend Snippet: TP53 mutation status in select ovariancancer cell lines and THBS1 mRNA and protein expression . The three ovarian cancer cell lines with wt TP53 demonstrated higher levels of THBS1 mRNA, but lower relative protein expression. Conversely, mutant TP53 cell lines had lower levels of THBS1 mRNA expression and higher levels of relative protein expression. TP53 missense mutation ; wt TP53 gene .

Article Snippet: Primers and probes for THBS1 (Hs00170236_m1) and GAPDH (human 402869) were obtained from Applied Biosystems (Foster City, CA, USA).

Techniques: Mutagenesis, Expressing

Journal: Frontiers in Oncology

Article Title: TP53 Status is Associated with Thrombospondin1 Expression In vitro

doi: 10.3389/fonc.2013.00269

Figure Lengend Snippet: THBS1 mRNA expression in ovarian cancer cell lines following radiation and hypoxia treatment . Induction of THBS1 transcription in the parent A2780wt TP53 cells and A2780m TP53 cells following radiation treatment (A) ; and hypoxia exposure (B) . After treatment with radiation, the A2780wtTP53 cells demonstrated a 3.6-fold increase at 24 h while the A2780m TP53 cells had a 4.5-fold increase at 24 h and a 9.5-fold increase at 48 h. There was a 3.4-fold greater increase in THBS1 levels at 48 h in the A2780m TP53 cell line compared to wild type. There was an approximately fourfold increase in THBS1 levels in the A2780wt cells at 8 and 24 h. In the A2780m there was a 4.6-fold increase at 8 h, and a 2.8-fold increase at 24 h. Controls ; radiated A2780wt TP53 cells ; radiated A2780m TP53 cells ; hypoxia treated A2780wt TP53 cells ; and hypoxia treated A2780m TP53 cells .

Article Snippet: Primers and probes for THBS1 (Hs00170236_m1) and GAPDH (human 402869) were obtained from Applied Biosystems (Foster City, CA, USA).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: TP53 Status is Associated with Thrombospondin1 Expression In vitro

doi: 10.3389/fonc.2013.00269

Figure Lengend Snippet: Possible mechanisms of TP53 regulation of THBS1 expression . Wild-type (wt) TP53 may bind to the THBS1 promoter resulting in gene transcription. Alternatively, wt TP53 is normally degraded and expressed at low levels allowing for a secondary factor to bind to the promoter site. Missense p53 protein can bind with wt p53 protein and prevent it from forming homotetramers and/or interacting with DNA, or if it does interact with DNA, the presence of the mutant protein may impede interaction with other secondary factors required to drive induction of transcription. In rare cases, upon mutation the wt p53 DNA-binding activity is lost and the p53 target regions are vulnerable to de novo cytosine methylation (Me) that inhibits transcription. Null TP53 mutations lead to complete loss of function of the gene and abnormal p53 fragments that are degraded. This may allow for a secondary factor to bind and induce THBS1 transcription.

Article Snippet: Primers and probes for THBS1 (Hs00170236_m1) and GAPDH (human 402869) were obtained from Applied Biosystems (Foster City, CA, USA).

Techniques: Expressing, Mutagenesis, Binding Assay, Activity Assay, Methylation

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: PRSS21/Testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4

doi: 10.1007/s00109-019-01763-3

Figure Lengend Snippet: Angiogenic genes altered by constitutive testisin expression in ES-2-Luc-TsWT cells compared to ES-2-Luc-Ctl Cells *

Article Snippet: ​ Genes Upregulated ≥ 2-Fold Gene Symbol Assay ID ΔC T ES-Luc-Ctl ΔC T ES-Luc-TsWT Fold Change PECAM1 Hs00169777_m1 15.98 13.96 4.05 CDH5 Hs00174344_m1 8.03 6.98 2.07 FST Hs00246256_m1 7.05 6.00 2.07 EDIL3 Hs00174781 _m1 14.01 12.98 2.04 IL12A Hs00168405_m1 10.01 8.98 2.04 TNF Hs00174128_m1 18.05 17.03 2.03 CXCL2 Hs00601975_m1 7.99 6.97 2.03 THBS1 Hs00962914_m1 8.00 6.98 2.03 Genes Downregulated ≥ 2-Fold Gene Symbol Assay ID ΔC T ES-Luc-Ctl ΔC T ES-Luc-TsWT Fold Change ANGPTL4 Hs01101127_m1 13.07 16.02 7.76 PDGFB Hs00234042_m1 12.01 14.03 4.05 ANGPTL2 Hs00765775_m1 14.02 16.02 4.01 KIT Hs00174029_m1 13.00 15.00 4.01 PDGFRB Hs00387364_m1 13.05 15.05 4.01 COL18A1 Hs00181017_m1 9.03 11.03 3.99 THBS2 Hs01568063_m1 16.05 18.01 3.90 TNNI1 Hs00913333_m1 17.03 18.28 2.38 COL4A3 Hs01022527_m1 14.89 16.13 2.36 EPHB2 Hs00362096_m1 6.96 8.01 2.06 FGF4 Hs00173564_m1 18.99 20.03 2.06 ITGA4 Hs00168433_m1 9.00 10.03 2.04 TYMP Hs00157317_m1 15.00 16.02 2.03 HSPG2 Hs00194179_m1 9.01 10.02 2.02 PTN Hs00383235_m1 12.03 13.04 2.02 TIE1 Hs00178500_m1 5.02 6.03 2.02 HEY1 Hs00232618_m1 12.02 13.02 2.00 ITGB3 Hs01001469_m1 7.01 8.01 2.00 FBLN5 Hs00197064_m1 19.00 20.00 2.00 ANGPT2 Hs00169867_m1 11.98 12.98 2.00 Open in a separate window * Total RNA was isolated from ES-2-Luc-TsWT and ES-2-Luc-Ctl and analyzed by TaqMan Angiogenesis Signature Array (ThermoFisher) according to the manufacturers’ instructions.

Techniques: Expressing

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A and B) Images of optic nerve sections showing CTB-labeled axons (grey) in C57BL/6J mice injected with either (A) AAV-THBS1 or (B) AAV-GFP. Asterisks, lesion site. Scale bars, 100 μm.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Labeling, Injection

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A) A schematic of THBS1 mutants investigated. All constructs contain the N-terminal signal peptide and have a C-terminal HA tag. Laminin G domain (LamG), oligomerization coiled coil (CC) domain, von Willebrand complex like domain (vWC), thrombospondin type 1 repeat domain (TSR1), epidermal growth factor-like repeat domains (EGF), type 3 repeat domain (TSR3), and the thrombospondin C-terminal domain (CTD). THBS4 is shown for comparison to THBS1.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Construct

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A) Images of optic nerve section showing GFP-labeled axons (green) from HB9:GFP;Bax−/− mice and CTB (magenta) following injection with AAV-THBS1 and optic nerve crush. Asterisks, lesion site.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Labeling, Injection

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: shRNA, Recombinant, Multiplex Assay, Clone Assay, Software