Journal: Oncotarget

Article Title: In vivo RNAi screen identifies NLK as a negative regulator of mesenchymal activity in glioblastoma

doi:

Figure Lengend Snippet: A. Schematic representation of in vivo RNAi screening. Patient-derived GBM cells are transduced with the shRNA library pool and injected into the mice brains. Tumors were harvested and shRNAs were PCR amplified and deep sequenced to identify candidate “hits”. B. Analysis of the shRNAs recovered from our RNAi screening. Data are normalized to the Control population and plotted in Log2 scale. Red box indicates enriched shRNAs. Enrichment of PTEN shRNAs in GBM 2 tumors (PTEN WT) compared to the GBM 3 tumors (PTEN deletion). C. A selection criteria for candidate hits from the RNA interference screen. D. Candidate “hits” from the screen. E. Immunohistochemistry of NLK on TissueMicroArray (TMA) containing 88 GBM samples and 32 normal brain samples. Scale bar, 100 μm. F. Oncomine microarray data analysis for NLK expression in glioblastoma versus normal brain tissues. ( p < 0.001, n = 525) G. Analysis of REMBRANDT public dataset on glioma patient survival in accordance with NLK high and NLK low expressions ( n = 90 each). H. Representative confocal microscopy images of immunofluorescence (IF) staining of NLK, Sox2, and GFAP in normal neural progenitor cells (NPC), differentiated NPC, and GBM cells. Scale bar, 100 μm. I. Immunoblots of NLK in patient-derived GBM cells transduced with Control or shRNA NLK. J. Kaplan-Meier survival curves of Control vs shRNA NLK. K–L , Schematic representation of dual-color competition assay in vivo . K A total of 50,000 cells from a 1:1 mixture of RFP-labeled shControl cells (red) and GFP-labeled shNLK cells (green) were implanted into mouse brains. L Immunofluorescence images of cryo-sectioned mouse brains. Scale bar, 500 μm. Bar graph represents the number of GFP and RFP positive cells that were counted in different spots of the tumor that were selected randomly. (+ SD, n = 4).

Article Snippet: We have generated a shRNA pool directed against these gene sets by selecting individual shRNA lentiviral clones from Cold Spring Harbor shRNA libraries.

Techniques: In Vivo, Derivative Assay, Transduction, shRNA, Injection, Amplification, Control, Selection, Immunohistochemistry, Microarray, Expressing, Confocal Microscopy, Immunofluorescence, Staining, Western Blot, Competitive Binding Assay, Labeling

Journal: Oncogene

Article Title: Transducin (β)-like 1 X-linked receptor 1 promotes gastric cancer progression via the ERK1/2 pathway

doi: 10.1038/onc.2016.352

Figure Lengend Snippet: Expression of TBL1XR1 in GC tissues and cell lines. ( a ) QRT-PCR analyses of TBL1XR1 mRNA expression in GC and patient-matched normal tissues ( n= 31). ( b ) Histogram displaying the relative mRNA expression of TBL1XR1 in 31 GC tissues. ( c ) Immunohistochemical staining of TBL1XR1 in GC tissues. Representative images of TBL1XR1 staining in surgical specimens from normal tissues, intestinal GC tissues and diffuse GC tissues (magnification: × 200). ( d ) Histogram shows the number of TBL1XR1 positive cells in gastric tumour tissues. ( e ) Cumulative survival curve of GC patients. ( f ) Immunofluorescence shows the nuclear localization of TBL1XR1 in GC tissues and paired normal tissues (magnification: × 200). ( g ) Histogram displaying the relative mRNA expression of TBL1XR1 in seven gastric tumour cell lines (SGC7901, MKN45, NCI-N87, BGC823, AGS, MKN28 and SNU-1) and one immortalized normal gastric epithelial cell line (GES-1). ( h ) The expression of TBL1XR1 protein in gastric tumour cell lines and immortalized normal gastric epithelial cell line (GES-1) was examined by western blot analysis. *P <0.05, **P <0.01 , ***P <0.001.

Article Snippet: TBL1XR1 shRNA sequences and negative control sequences were designed and synthesized by Genechem (Genechem Co. Ltd., Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Immunofluorescence, Western Blot

Journal: Oncogene

Article Title: Transducin (β)-like 1 X-linked receptor 1 promotes gastric cancer progression via the ERK1/2 pathway

doi: 10.1038/onc.2016.352

Figure Lengend Snippet: Relationship between TBL1XR1 expression level and clinicopathologic variables in 134 GC tissues

Article Snippet: TBL1XR1 shRNA sequences and negative control sequences were designed and synthesized by Genechem (Genechem Co. Ltd., Shanghai, China).

Techniques: Expressing, Immunostaining

Journal: Oncogene

Article Title: Transducin (β)-like 1 X-linked receptor 1 promotes gastric cancer progression via the ERK1/2 pathway

doi: 10.1038/onc.2016.352

Figure Lengend Snippet: The effects of TBL1XR1 on tumour cell proliferation and apoptosis. ( a ) Western blot analyses of TBL1XR1 expression in NCI-N87 and SGC7901 cells underwent transient transfection of TBL1XR1 shRNA (sh#1, sh#2, sh#3 and sh#4). ( b ) TBL1XR1 protein expression in NCI-N87 and SGC7901 cells transfected with TBL1XR1-shRNA (sh#3) was confirmed by western blot analysis. ( c ) TBL1XR1 protein expression in BGC823 and MKN45 cells transfected with TBL1XR1-constructed plasmid. ( d ) The effect of TBL1XR1 knockdown on NCI-N87 and SGC7901 cell proliferation was analysed by CCK8 assay. ( e ) The effect of TBL1XR1 overexpression on BGC823 and MKN45 cell proliferation was analysed by CCK8 assay. ( f ) and ( g ) Knockdown of TBL1XR1 induced the apoptosis of NCI-N87 cells. ( h ) and ( i ) Overexpression of TBL1XR1 promoted the clone formation of BGC823 cells. Data are representative of three independent experiments (mean±s.d.). * P <0.05, ** P <0.01.

Article Snippet: TBL1XR1 shRNA sequences and negative control sequences were designed and synthesized by Genechem (Genechem Co. Ltd., Shanghai, China).

Techniques: Western Blot, Expressing, Transfection, shRNA, Construct, Plasmid Preparation, Knockdown, CCK-8 Assay, Over Expression

Journal: Oncogene

Article Title: Transducin (β)-like 1 X-linked receptor 1 promotes gastric cancer progression via the ERK1/2 pathway

doi: 10.1038/onc.2016.352

Figure Lengend Snippet: TBL1XR1 enhances the migratory and invasive ability of GC cells. ( a ), ( c ) and ( e ) Effects of knockdown and overexpression of TBL1XR1 on GC cell wound healing, migration and invasion was measured and representative images of distance (mm) of wound healing, migrated and invaded cells (magnification: × 100) are shown. ( b ), ( d ) and ( f ) Histogram shows the relative distance (mm) (magnification: × 100) of wound healing and the number of migrated and invaded cells. Five random fields were selected for statistical analysis. ( g ) and ( i ) The effects of knockdown and overexpression of TBL1XR1 on EMT markers was determined by western blot analysis. Densitometry shows relative protein expression normalized for GAPDH. ( h ) and ( j ) Densitometric analysis shows the effects of knockdown and overexpression of TBL1XR1 on EMT markers of GC cells. Data are shown as mean±s.d. of three independent experiments. * P <0.05, **P <0.01.

Article Snippet: TBL1XR1 shRNA sequences and negative control sequences were designed and synthesized by Genechem (Genechem Co. Ltd., Shanghai, China).

Techniques: Knockdown, Over Expression, Migration, Western Blot, Expressing

Journal: Oncogene

Article Title: Transducin (β)-like 1 X-linked receptor 1 promotes gastric cancer progression via the ERK1/2 pathway

doi: 10.1038/onc.2016.352

Figure Lengend Snippet: TBL1XR1 enhances GC cell proliferation, migration and invasion via the ERK1/2 signalling pathway. ( a ) The expression of TBL1XR1 and pERK1/2 in 134 pairs human GC tissues were assessed by IHC. The representative images show the expression of TBL1XR1 and pERK1/2 in intestinal and diffuse type of GC (scale bar=100 μm). ( b ) Spearman correlation was used to analyse the correlation between TBL1XR1 and pERK1/2 expression in 134 pairs of GC tissues. ( c ) The expression of pERK1/2, ERK1/2 and TBL1XR1 in four GC cell lines were determined by western blot analysis. ( d ) ERK1/2 specific inhibitor U0126 (20 μ M ) suppressed the proliferation of NCI-N87 and SGC7901 cells. ( e ) and ( f ) U0126 (20 μM) inhibited the migratory and invasive ability of NCI-N87 and SGC7901 cells (scale bar=100 μm). ( g ) U0126 (20 μ M ) inhibited the phosphorylation of ERK1/2 and EMT changes in NCI-N87 cells and SGC7901 cells. ( h ) The pERK1/2 level in NCI-N87/TBL1XR1-shRNA and SGC7901/TBL1XR1-shRNA cells was determined by western blot analysis. ( i ) The effect of TBL1XR1 overexpression on pERK1/2 level in BGC823 and MKN45 cells. ( j ) BGC823/TBL1XR1 and MKN45/TBL1XR1 cell proliferation were measured in the presence of U026 (20 μ M ). ( k ) and ( l ) The effect of U0126 on the migratory and invasive ability of BGC823/TBL1XR1 and MKN45/TBL1XR1 cells was analysed and representative images are shown (magnification: × 100, scale bar=100 μm). ( m ) The effect of U0126 (20 μ M ) on the EMT changes of BGC823/TBL1XR1 and MKN45/TBL1XR1 cells was analysed by western blot analysis. Densitometry shows relative protein expression normalized for GAPDH. Data are representative of three independent experiments (mean±s.d.). * P <0.05, ** P <0.01.

Article Snippet: TBL1XR1 shRNA sequences and negative control sequences were designed and synthesized by Genechem (Genechem Co. Ltd., Shanghai, China).

Techniques: Migration, Expressing, Western Blot, Phospho-proteomics, shRNA, Over Expression

Journal: Oncogene

Article Title: Transducin (β)-like 1 X-linked receptor 1 promotes gastric cancer progression via the ERK1/2 pathway

doi: 10.1038/onc.2016.352

Figure Lengend Snippet: The activation of ERK1/2 induced by TBL1XR1 is mediated via the β-catenin signalling pathway. ( a ) The phosphorylation levels of β-catenin (p-β-catenin) and ERK1/2 in NCI-N87/TBL1XR1-shRNA and SGC7901/TBL1XR1-shRNA cells were detected by western blot analysis. ( b ) The phosphorylation levels of β-catenin and ERK1/2 in BGC823/TBL1XR1 and MKN45/TBL1XR1 cells were detected by western blot analysis. ( c ) The phosphorylation level of ERK1/2 and EMT in BGC823/TBL1XR1 and MKN45/TBL1XR1 cells treated with XAV939 were detected by western blot analysis (10 μ M ). ( d ) The effect of U0126 (20 μ M ) on phosphorylation of β-catenin in NCI-N87 and SGC7901 cells was analysed by western blot analysis. Densitometry shows relative protein expression.

Article Snippet: TBL1XR1 shRNA sequences and negative control sequences were designed and synthesized by Genechem (Genechem Co. Ltd., Shanghai, China).

Techniques: Activation Assay, Phospho-proteomics, shRNA, Western Blot, Expressing

Journal: Oncogene

Article Title: Transducin (β)-like 1 X-linked receptor 1 promotes gastric cancer progression via the ERK1/2 pathway

doi: 10.1038/onc.2016.352

Figure Lengend Snippet: The activation of ERK1/2 induced by TBL1XR1 is mediated via the β-catenin/MMP7/EGFR signalling pathway. ( a ) and ( b ) The expression levels of TBL1XR1, p-β-catenin, MMP7, pEGFR and pERK1/2 in NCI-N87/TBL1XR1-shRNA cells ( a ) and BGC823/TBL1XR1 cells ( b ) were detected by western blot analysis. ( c ) and ( d ) The expression levels of TBL1XR1, p-β-catenin, MMP7, pEGFR and pERK1/2 in NCI-N87 cells ( c ) and BGC823/TBL1XR1 cells ( d ) in the presence of XAV939 (10 μ M ), Batimastat (10 μ M ), Afatinib (5 μ M ) and U0126 (20 μ M ) were detected by western blot analysis. Densitometry shows relative protein expression.

Article Snippet: TBL1XR1 shRNA sequences and negative control sequences were designed and synthesized by Genechem (Genechem Co. Ltd., Shanghai, China).

Techniques: Activation Assay, Expressing, shRNA, Western Blot

Journal: Oncogene

Article Title: Transducin (β)-like 1 X-linked receptor 1 promotes gastric cancer progression via the ERK1/2 pathway

doi: 10.1038/onc.2016.352

Figure Lengend Snippet: TBL1XR1 promotes subcutaneous tumour growth and peritoneal dissemination in nude mice. NCI-N87/nc-shRNA, NCI-N87/TBL1XR1-shRNA, BGC823/vector and BGC823/TBL1XR1 cells were subcutaneously or intraperitoneally injected into nude mice. Tumour volume was monitored twice a week and all mice were sacrificed under general anesthesia 6 weeks after injection. ( a ) and ( b ) Representative images of tumour-bearing mice and tumour mass. ( c ) and ( d ) Tumour growth curves. ( e ) and ( f ) Average tumour weight of each group ( N= 5). ( g ) Immunohistochemistry analyses of TBL1XR1, pERK1/2, Ki67, E-cadherin and ZEB2 protein expression in xenograft tumours. ( h–j ) NCI-N87/TBL1XR1-shRNA, NCI-N87/nc-shRNA, BGC823/TBL1XR1 and BGC823/vector cells were intraperitoneally transplanted into the nude mice, the volume of ascites and the number of peritoneal nodules from each mouse were measured. Macroscopic images of the peritoneal disseminations are shown and red arrows in the images indicate tumours developing peritoneal invasion and dissemination. ( k ) and ( l ) Average of the peritoneal tumour nodules developed by NCI-N87/TBL1XR1-shRNA, NCI-N87/nc-shRNA, BGC823/TBL1XR1 and BGC823/vector cells was quantified. Data are representative of three independent experiments (mean±s.d.). * P <0.05, ** P <0.01.

Article Snippet: TBL1XR1 shRNA sequences and negative control sequences were designed and synthesized by Genechem (Genechem Co. Ltd., Shanghai, China).

Techniques: shRNA, Plasmid Preparation, Injection, Immunohistochemistry, Expressing

Journal: Oncogene

Article Title: Transducin (β)-like 1 X-linked receptor 1 promotes gastric cancer progression via the ERK1/2 pathway

doi: 10.1038/onc.2016.352

Figure Lengend Snippet: Schematic model of TBL1XR1-promoting ERK activation and gastric cancer progression. GC cell-derived TBL1XR1 promotes the phosphorylation of ERK1/2 via activating β-catenin/MMP7/EGFR, which facilitates the EMT, migration, invasion and metastatic potential of GC cells.

Article Snippet: TBL1XR1 shRNA sequences and negative control sequences were designed and synthesized by Genechem (Genechem Co. Ltd., Shanghai, China).

Techniques: Activation Assay, Derivative Assay, Phospho-proteomics, Migration