Journal: Stem cells (Dayton, Ohio)

Article Title: FUS-CHOP fusion protein expression coupled to p53 deficiency induces liposarcoma in mouse but not in human adipose-derived mesenchymal stem/stromal cells.

doi: 10.1002/stem.571

Figure Lengend Snippet: Figure 1. Characterization of FUS-CHOP expressing mouse ASCs. (A): Reverse transcription polymerase chain reaction confirming the expression of FUS-CHOP in wt and p53/ ASCs at mRNA level. GAPDH was used as housekeeping gene. (B): Western blotting showing the expression of FUS- CHOP using an anti-GADD153/CHOP antibody and the p53 status of the indicated mASCs. b-actin was used as loading control. (C): Immunophenotypic profile of the indicated ASC genotypes analyzed by flow cytometry. Representative dot plots are shown for Sca-1, CD29, CD44, CD14, CD11b, and CD45. Filled lines represent the irrelevant isotypes. (D): Adipogenic (oil red staining, upper panels) and osteogenic (Alizarin red staining, bottom panels) differentiation potential of ASCs with the distinct genotypes indicated. Inset images represent negative controls of differentiation. Abbreviations: FC, FUS-CHOP; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, mASC, mouse adipose-derived mesenchymal stem/stromal cell; wt, wild type.

Article Snippet: Depletion of p53 was achieved by transduction with lentiviral particles carrying a p53-shRNA expression vector (pLVUH-shp53; Addgene plasmid 11,653 [24]).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Control, Cytometry, Staining, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: FUS-CHOP fusion protein expression coupled to p53 deficiency induces liposarcoma in mouse but not in human adipose-derived mesenchymal stem/stromal cells.

doi: 10.1002/stem.571

Figure Lengend Snippet: Figure 2. Characterization of tumors developed by mASC-p53-GFP and mASC-p53-FC. (A): Summary table indicating tumor incidence, la- tency, and histological analysis of tumors developed in immunedeficient Nonobese diabetic/severe combined immunodeficient NOD.Cc-Prkdcscid

Article Snippet: Depletion of p53 was achieved by transduction with lentiviral particles carrying a p53-shRNA expression vector (pLVUH-shp53; Addgene plasmid 11,653 [24]).

Techniques:

Journal: Stem cells (Dayton, Ohio)

Article Title: FUS-CHOP fusion protein expression coupled to p53 deficiency induces liposarcoma in mouse but not in human adipose-derived mesenchymal stem/stromal cells.

doi: 10.1002/stem.571

Figure Lengend Snippet: Figure 3. Characterization of ex vivo-established transformed mASC lines from FUS-CHOP-expressing tumors. (A): Reverse transcription poly- merase chain reaction showing the expression of FUS-CHOP at mRNA level in the indicated tumor-derived cell lines (T-AD-p53-FC #1 to #5). GAPDH was used as a housekeeping gene. (B): Western blotting confirming the expression of FUS-CHOP protein using an anti-GADD153/CHOP antibody. b-actin was used as loading control. (C): Summary table indicating tumor incidence, latency, and histological analysis of tumors developed in NOD/SCID IL2Rc/ mice inoculated with the indicated ASC lines. (*) The average number of days needed to observe a tumor diameter of 8 mm. (D): H&E and oil red O staining of tumors arising in mice inoculated with the indicated ASC lines. Abbreviations: FC, FUS-CHOP; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; mASC, mouse adipose-derived mesenchymal stem/stromal cells.

Article Snippet: Depletion of p53 was achieved by transduction with lentiviral particles carrying a p53-shRNA expression vector (pLVUH-shp53; Addgene plasmid 11,653 [24]).

Techniques: Ex Vivo, Transformation Assay, Expressing, Reverse Transcription, Derivative Assay, Western Blot, Control, Staining

Journal: Stem cells (Dayton, Ohio)

Article Title: FUS-CHOP fusion protein expression coupled to p53 deficiency induces liposarcoma in mouse but not in human adipose-derived mesenchymal stem/stromal cells.

doi: 10.1002/stem.571

Figure Lengend Snippet: Figure 4. Signaling pathways altered by the expression of FUS-CHOP in mASCs. (A–C): After gene expression microarray analysis, the groups of genes differentially expressed (p value <0.01; expression more than two fold up or down) in mASC-p53-GFP or mASC-p53-FC versus mASC-wt-GFP were compared and the lists of pathways significantly altered were generated using the Ingenuity Pathways Analysis 8 software. (A): Venn diagram showing the number of genes commonly and specifically altered in mASC-p53-GFP and/or mASC-p53-FC cells. (B): List of significantly modulated pathways (p < 0.05) generated with the genes commonly altered in both mASC-p53-GFP and mASC-p53-FC cells. Only a selection of relevant pathways is shown (see Supporting Information Table 2 for the complete list of pathways). (C): List of significantly altered pathways (p < 0.05) generated with the genes specifically altered in mASC-p53-FC cells. Arrowheads point to key pathways that are likely to contribute to liposarcoma formation. (D): Expression of adipogenic regulators in mASC-wt-FC and mASC-p53-FC cells relative to mASC-wt-GFP cells. Differentially expressed genes were obtained by microarray analysis (p value <0.01; expression more than twofold up or down). þ/ indicates genes upregulated/dowregulated up to four times; þþ/ indicates genes upregulated/dowregulated more than four times. ‘‘o’’ represents genes not significantly altered. (E): Western blotting showing the level of expression of PPARc and C/EBPa in cell lines derived from tumors arisen from mASC-p53-FC cells (T-AD-p53-FC-3) and mASC-p53-GFP cells (T-AD-p53-GFP-1). Total extracts from murine white adipose tissue was included as a positive control and b-actin was used as loading control. Abbreviations: AKT, v-akt murine thymoma viral onco- gene homolog; CNTF, ciliary neurotrophic factor; C/EBPa, CCAAT/enhancer binding protein a; FC, FUS-CHOP; FGF, fibroblast growth factor; GFP, green fluorescent protein; GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; mASC, mouse adipose-derived mes- enchymal stem/stromal cells; PAK, p21 protein (Cdc42/Rac)-activated kinase; PDGF, platelet-derived growth factor; PPARc, peroxisome prolifer- ator-activated receptor c; PTEN, phosphatase and tensin homolog; WAT, white adipose tissue.

Article Snippet: Depletion of p53 was achieved by transduction with lentiviral particles carrying a p53-shRNA expression vector (pLVUH-shp53; Addgene plasmid 11,653 [24]).

Techniques: Protein-Protein interactions, Expressing, Gene Expression, Microarray, Generated, Software, Selection, Western Blot, Derivative Assay, Positive Control, Control, Binding Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: FUS-CHOP fusion protein expression coupled to p53 deficiency induces liposarcoma in mouse but not in human adipose-derived mesenchymal stem/stromal cells.

doi: 10.1002/stem.571

Figure Lengend Snippet: Figure 5. Generation of FUS-CHOP-expressing wt or p53-deficient human adipose-derived mesenchymal stem/stromal cells (hASCs). (A): hASCs were transduced with lentiviral particles expressing FUS-CHOP or GFP. The GFPþ population was enriched to purify FUS-CHOP-expressing hASCs. Subsequently, p53 was depleted from some of the cultures to obtain the following genotypes: GFP, FC, p53-GFP, and p53-FC. Representative images of GFPþ hASCs and GFP analysis by flow cytometry are shown. (B): RT-PCR (upper panel) and Western blot (lower panel) showing FC expression at RNA and protein levels in FC-transduced hASCs. GAPDH and FUS were respectively used as loading controls. (C): Western blotting showing p53 and p21 protein levels after transduction of GFP- and FC-expressing hASCs with lentiviral particles-expressing p53 shRNA. Cells were treated with or without 1 lM CPT for 24 hours. b-actin was used as loading control. (D): Phase-contrast morphology, adipogenic (oil red staining), and osteogenic (Alizarin red staining) differentiation potential of the distinct hASCs. Abbreviations: CPT, campthotecin; FC, FUS-CHOP; GAPDH, glyc- eraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; RT-PCR, reverse transcription polymerase chain reaction; WB, western blot.

Article Snippet: Depletion of p53 was achieved by transduction with lentiviral particles carrying a p53-shRNA expression vector (pLVUH-shp53; Addgene plasmid 11,653 [24]).

Techniques: Expressing, Derivative Assay, Transduction, Cytometry, Reverse Transcription Polymerase Chain Reaction, Western Blot, shRNA, Control, Staining, Reverse Transcription, Polymerase Chain Reaction

Journal: Stem cells (Dayton, Ohio)

Article Title: FUS-CHOP fusion protein expression coupled to p53 deficiency induces liposarcoma in mouse but not in human adipose-derived mesenchymal stem/stromal cells.

doi: 10.1002/stem.571

Figure Lengend Snippet: Figure 6. In vitro growth properties of FUS-CHOP-expressing wt or p53-deficient hASCs. (A): Cumulative population doublings of the indicated cultures. The time of p53 depletion (dashed arrow), senescence entry (blue arrows), and a representative image of a senescence-associated b-galacto- sidase activity staining are shown. (B): Cell cycle analysis including BrdU labeling of S-phase. (C): Colony-forming ability. (D): Soft agar assay showing no anchorage-independent growth of either wt or p53-deficient hASCs-expressing FUS-CHOP. HeLa cells and T-AD-p53-FC #4 were used as a positive control. (E): Summary table indicating the inability of hASC-p53-GFP and hASC-p53-FC cells inoculated in NOD/SCID IL2Rc/

Article Snippet: Depletion of p53 was achieved by transduction with lentiviral particles carrying a p53-shRNA expression vector (pLVUH-shp53; Addgene plasmid 11,653 [24]).

Techniques: In Vitro, Expressing, Activity Assay, Staining, Cell Cycle Assay, Labeling, Soft Agar Assay, Positive Control

Journal: Stem cells (Dayton, Ohio)

Article Title: FUS-CHOP fusion protein expression coupled to p53 deficiency induces liposarcoma in mouse but not in human adipose-derived mesenchymal stem/stromal cells.

doi: 10.1002/stem.571

Figure Lengend Snippet: Figure 7. Signaling pathways altered by the expression of FUS-CHOP in hASCs. (A): Heat map diagram summarizing the microarray gene expression changes (p value < .05; expression more than twofold up or down) in hASC-wt-FC compared with hASC-wt-GFP cells. Genes involved in Wnt/bcatenin (red), endothelin-1 (blue), G-protein-coupled receptor (green), and other relevant signaling pathways (black) are indi- cated. (B–D): Lists containing the significantly (p < .05) altered pathways generated using Ingenuity Pathway Analysis software with the genes differentially expressed in hASC-wt-FC versus hASC-wt-GFP cells (B), hASC-p53-FC versus hASC-wt-GFP cells (C), and mASC-p53-FC versus mASC-wt-GFP cells (D). Only a selection of relevant pathways is shown in ([D]; see Supporting Information Table 2 for the complete list of pathways). Abbreviations: AKT, v-akt murine thymoma viral oncogene homolog; ATM, ataxia telangiectasia mutated; BRCA1, breast cancer 1, early onset; CHK, CHK checkpoint homolog; FC, FUS-CHOP; FGF, fibroblast growth factor; GFP, green fluorescent protein; GNRH, gonadotro- pin-releasing hormone; hASC, human adipose-derived mesenchymal stem/stromal cell; HER-2, Human Epidermal growth factor Receptor; IL-1, interleukin-1; IRF, interferon regulatory factor; LPS, lipopolysaccharide; PDGF, platelet-derived growth factor; PKR, protein kinase R; PTEN, phosphatase and tensin homolog; RXR, retinoid X receptor; THOP1, thimet oligopeptidase 1; TNFR2, tumor necrosis factor receptor 2; TREM1, triggering receptor expressed on myeloid cells 1; VDR, vitamin D receptor; wt, wild type.

Article Snippet: Depletion of p53 was achieved by transduction with lentiviral particles carrying a p53-shRNA expression vector (pLVUH-shp53; Addgene plasmid 11,653 [24]).

Techniques: Protein-Protein interactions, Expressing, Microarray, Gene Expression, Generated, Software, Selection, Derivative Assay