Journal: Cell Death & Disease

Article Title: TGF- β is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion

doi: 10.1038/cddis.2014.395

Figure Lengend Snippet: TGF- β induces mesenchymal transdifferentiation in GBM cells that is associated with enhanced migratory and invasive capacity. ( a ) Western blot showing the activation of TGF- β pathway as indicated by the phosphorylation of SMAD2. ( b ) Phase contrast microscopy at × 10 magnification showing TGF- β -induced changes in cellular morphology in U87 and U251 cells associated with spindle-shaped morphology and a more scattered growth pattern. ( c ) Immunofluorescence analysis depicting enhanced expression of the mesenchymal markers Fibronectin and COL5A1 following TGF- β exposure of U87 and U251 cells; images obtained at × 20 magnification. ( d ) A representative wound healing assay showing enhanced migratory capacity in U251 cells following exposure to TGF- β compared with the untreated group. Quantification of the wound closure capacity after 24 h of wound healing time ( n =3) is shown in ( e ) (*** P <0.001 U251 without TGF- β versus with TGF- β for their respective time points). ( f ) A representative Transwell collagen assay showing Coomassie blue-stained cells on the insert membranes, demonstrating enhanced invasive capacity following exposure to TGF- β when compared with untreated counterparts. Chemoattractants were 10% FCS or 0.1% FCS supplemented with EGF (50 ng/ml). Quantified data depicted in ( g ) and the bars represent the mean of in general three independent experiments measured in triplicate±S.E.M. (* P <0.05, ** P <0.01 and *** P <0.001, U87 without versus with TGF- β for their respective groups; # P <0.05, ### P <0.001, U87 10% FCS or EGF versus 0.1% FCS)

Article Snippet: U87 cells untreated or pretreated with TGF- β (T) for 96 h in the presence or absence of the TGF- β inhibitor (I) A8301 (Axon Medchem) added 2 h before TGF- β addition were prepared for intracranial injection in NOD SCID gamma mice (NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ)/NSG mice) obtained as inbred strains from the Central Animal Facility (Groningen, The Netherlands).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Microscopy, Immunofluorescence, Expressing, Wound Healing Assay, Collagen Assay, Staining

Journal: Cell Death & Disease

Article Title: TGF- β is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion

doi: 10.1038/cddis.2014.395

Figure Lengend Snippet: TGF- β signaling inhibitor A8301 prevents TGF- β -induced mesenchymal shift and enhanced invasive ability of GBM cells. ( a ) Western blots showing inhibition of TGF- β -induced SMAD2 phosphorylation and expression of the mesenchymal marker Fibronectin by A8301 (0.5 μ M). Representative blots are shown of n =3. ( b ) Phase contrast microscopic pictures at × 10 magnification of U87 and U251 cells treated with TGF- β in the presence or absence of A8301 in comparison with the untreated controls. The inhibitor prevented the phenotypic shift induced by TGF- β . ( c ) Wound healing assays showing reduced migration of TGF- β -treated U251 cells in the presence of A83-01. Quantification of the wound closure capacity ( n =3, *** P <0.001) is shown in ( d ). ( e ) Transwell collagen assays showing reduced invasion of U87 cells toward 10% FCS following the addition of A83-01. Membranes were fixed and evaluated for cell numbers. A representative picture of the membranes showing invading U87 cells is shown in ( e ) and quantification of invasion assay is shown in ( f ) in which bars represent the mean of in general three independent experiments±S.E.M. (** P <0.01, *** P <0.001, U87 for 10% FCS group; ## P <0.01, for U87 10% FCS versus 0.1% FCS)

Article Snippet: U87 cells untreated or pretreated with TGF- β (T) for 96 h in the presence or absence of the TGF- β inhibitor (I) A8301 (Axon Medchem) added 2 h before TGF- β addition were prepared for intracranial injection in NOD SCID gamma mice (NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ)/NSG mice) obtained as inbred strains from the Central Animal Facility (Groningen, The Netherlands).

Techniques: Western Blot, Inhibition, Phospho-proteomics, Expressing, Marker, Comparison, Migration, Invasion Assay

Journal: Cell Death & Disease

Article Title: TGF- β is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion

doi: 10.1038/cddis.2014.395

Figure Lengend Snippet: TGF- β -treated U87 cells show distinct tumor morphology and enhanced invasion after intracranial injection in NSG mice when compared with untreated cells, and can be prevented by cotreatment with A8301. ( a ) Overview of the experimental conditions used for intracranial transplantation. TGF- β (T) and A8301 (I), together with tumor take and the occurrence of neurological symptoms. ( b ) Immunohistochemical staining using the indicated markers of U87 xenografts. ( c ) Representative H&E staining of intracranial tumors derived from untreated U87 cells (T−I−), TGF- β- exposed cells (T+I−),TGF- β /A8301-exposed cells (T+I+) and cells exposed to A8301 alone (T−I+). TGF- β -treated tumor grafts showed cells with a more elongated/spindle-shaped cell morphology and appear more loosely packed when compared with nontreated, combined A8301 or A8301-alone-exposed cells. ( d ) Nestin-stained xenograft-normal brain parenchyma borders show enhanced invasion by groups or individual cells in TGF- β -stimulated U87-derived tumors (T+I−) when compared with the other conditions. ( e ) Expression of the mesenchymal marker Fibronectin was detected mainly in U87/TGF- β xenografts by immunohistochemistry. The T+I− tumors showed evidence of enhanced neutrophil infiltration (multinucleated cells, arrows) in H&E-stained specimens, and were also associated with increased vascularization and thicker basal membranes when compared with the other conditions visualized after Reticulin staining

Article Snippet: U87 cells untreated or pretreated with TGF- β (T) for 96 h in the presence or absence of the TGF- β inhibitor (I) A8301 (Axon Medchem) added 2 h before TGF- β addition were prepared for intracranial injection in NOD SCID gamma mice (NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ)/NSG mice) obtained as inbred strains from the Central Animal Facility (Groningen, The Netherlands).

Techniques: Injection, Transplantation Assay, Immunohistochemical staining, Staining, Derivative Assay, Expressing, Marker, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: TGF- β is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion

doi: 10.1038/cddis.2014.395

Figure Lengend Snippet: ZEB1 mediates TGF- β -induced mesenchymal transdifferentiation in GBM cells. ( a ) Western blots showing the effect of TGF- β administration on the expression of the indicated proteins, illustrating enhanced expression of the transcription factor ZEB1 in association with the upregulation of the mesenchymal markers Fibronectin, COL5A1 and MMP9 in U87 and U251 cells. The canonical Wnt signaling conferring transcription factor β -Catenin is also induced by TGF- β in U87 cells. Representative blots are shown of n =3. ( b ) A8301 prevents TGF- β -induced ZEB1 expression in GBM cells and inhibits β -Catenin accumulation in U87 cells. Consistently, induction of mesenchymal marker expression is prevented. Representative blots are shown of n =3. ( c ) Immunofluorescence microscopy ( × 40) showing accumulation and nuclear localization of ZEB1 in U87 cells. β -Catenin remains in the cytoplasm. ( d ) Phase contrast microscopy ( × 10) showing that TGF- β -induced acquisition of a mesenchymal morphology in U87 cells is prevented by siRNA-mediated silencing of ZEB1. Three different siRNAs directed against ZEB1 were used, of which the indicated two (siZEB1-I and siZEB1-II) were effective in blocking TGF- β -induced mesenchymal transdifferentiation when compared with siControl-transfected and mock-treated cells. ( e ) Expression levels were quantified with qRT-PCR and the bars represent the mean of in general three independent experiments measured in triplicate±S.E.M. (* P <0.05 and *** P <0.001, siZEB1-I and siZEB1-II versus scramble siRNA). ( f ) Wound healing assays showing reduced TGF- β -induced migratory activity upon silencing of ZEB1 using siZEB1-I or siZEB1-II. Quantified results of three independent results are shown in ( g ) (** P <0.01 and *** P <0.001, siZEB1-I and siZEB1-II versus scramble siRNA)

Article Snippet: U87 cells untreated or pretreated with TGF- β (T) for 96 h in the presence or absence of the TGF- β inhibitor (I) A8301 (Axon Medchem) added 2 h before TGF- β addition were prepared for intracranial injection in NOD SCID gamma mice (NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ)/NSG mice) obtained as inbred strains from the Central Animal Facility (Groningen, The Netherlands).

Techniques: Western Blot, Expressing, Marker, Immunofluorescence, Microscopy, Blocking Assay, Transfection, Quantitative RT-PCR, Activity Assay

Journal: Cell Death & Disease

Article Title: TGF- β is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion

doi: 10.1038/cddis.2014.395

Figure Lengend Snippet: Neurospheres demonstrate variability in acquiring mesenchymal properties following TGF- β treatment. ( a and b ) GG14 (PN) neurospheres exposed to TGF- β show enhanced proliferation as indicated by increased neurospheres sizes that could be prevented by the administration of A8301. This effect was not observed in GG16 (MES) neurospheres ( c and d ). Data represent the means±S.E.M. of 3 independent experiments where 30 neurospheres were included in each experiment (*** P <0.001). ( e ) Western blots showing the effect of TGF- β treatment on GG14 and GG16 in terms of the levels of the proneural markers (PDGFR- α , OLIG2) and mesenchymal marker (YKL40) along with ZEB1 and pSMAD2. ( f ) Representative Transwell membranes showing enhanced infiltrative capacity of TGF- β -treated GG14 neurospheres; quantification ( n =3) is shown in ( g ) (** P <0.01 and *** P <0.001, ### P <0.001, for GG14 control cells 0.1% FCS versus 10% FCS)

Article Snippet: U87 cells untreated or pretreated with TGF- β (T) for 96 h in the presence or absence of the TGF- β inhibitor (I) A8301 (Axon Medchem) added 2 h before TGF- β addition were prepared for intracranial injection in NOD SCID gamma mice (NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ)/NSG mice) obtained as inbred strains from the Central Animal Facility (Groningen, The Netherlands).

Techniques: Western Blot, Marker, Control

Journal: Cell Death & Disease

Article Title: TGF- β is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion

doi: 10.1038/cddis.2014.395

Figure Lengend Snippet: Local/regional mesenchymal transition detected in GBM patient material. ( a ) Immunohistochemical staining for pSMAD2, ZEB1 and YKL40 in consecutive sections detects overlapping expression patterns of pSMAD2, ZEB1 and YKL40 in perivascular areas in GBM patient tissue. ( b ) Model illustrating TGF- β -induced mesenchymal transition in GBM that is mediated by pSMAD2 and ZEB1. TGF- β may be produced by tumor cells, microglia or other stromal cells leading to a local induction of mesenchymal properties in GBM. This contributes to heterogeneity in GBM subtype

Article Snippet: U87 cells untreated or pretreated with TGF- β (T) for 96 h in the presence or absence of the TGF- β inhibitor (I) A8301 (Axon Medchem) added 2 h before TGF- β addition were prepared for intracranial injection in NOD SCID gamma mice (NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ)/NSG mice) obtained as inbred strains from the Central Animal Facility (Groningen, The Netherlands).

Techniques: Immunohistochemical staining, Staining, Expressing, Produced