Journal: Oncology Letters

Article Title: Inhibition of autophagy enhances apoptosis induced by bortezomib in AML cells

doi: 10.3892/ol.2020.12370

Figure Lengend Snippet: Downregulation of Beclin-1 enhances apoptosis and reduces cell viability induced by bortezomib in NB4 cells. Cells were infected with lentiviruses expressing shRNAs (non-targeting control or Beclin-1). Puromycin-resistant cells were pooled after each infection. (A) Cells transfected with the control shRNA and shBeclin-1 were treated with or without bortezomib (20 nM) for 24 h, and the expression levels of cleaved caspase-3, cleaved PARP, Bcl-2, p62 and Beclin-1, and LC3-I to LC3-II conversion were determined by western blotting. (B) Ratio of Bcl-2 and β-actin expression levels. Data are presented as the mean ± standard deviation of three independent repeats. *P<0.05 and **P<0.01. (C) Ratio of LC3-II and β-actin expression levels. Data are presented as the mean ± standard deviation of three independent repeats. **P<0.01. (D) Cells transfected with the control shRNA and shBeclin-1 were treated with bortezomib (20 nM) for 0 and 24 h, and cell viability was assessed using a water-soluble tetrazolium salts-8 assay. Data are presented as the mean ± standard deviation of three independent repeats. ***P<0.001. shRNA/sh, short hairpin RNA; CTRL, control; PARP, poly(ADP-ribose) polymerase.

Article Snippet: Lentiviral particles containing short hairpin RNA (shRNA/sh) Beclin-1 (cat. no. 131209BZ; 5′-CCGACTTGTTCCTTACGGAAA-3′) and shRNA negative control (shCTRL; cat. no. 131127CZ; 5′-TTCTCCGAACGTGTCACGTTTC-3′) expression vectors were obtained from Shanghai GenePharma Co., Ltd., and were then cloned into pGLV3/H1/GFP-Puro vector (Shanghai GenePharma Co., Ltd.).

Techniques: Infection, Expressing, Transfection, shRNA, Western Blot, Standard Deviation

Journal: Scientific reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro.

doi: 10.1038/srep43539

Figure Lengend Snippet: Figure 3. Tet2 knockdown decreases myoblast differentiation. (A) Knockdown of Tet2 by using shRNA. Upper: C2C12 cells were transfected with Tet2-specific shRNA plasmid (shTet2) or a control shRNA plasmid (shCtrl) and then selected with puromycin. The mRNA levels of Tet family genes or myoblast differentiation- associated genes in puromycin-selected cells were detected by qRT-PCR. Lower: the cells were induced to differentiation and the mRNA levels of Tets or myoblast differentiation-associated genes were detected 4 d after differentiation induction. Myog, myogenin; MyoM, myomaker. (B) Western blot confirmation of Tet2 protein decline in shRNA- or siRNA-mediated Tet2 knockdown cells. β-actin was used as an internal control. The expected Tet2 band (NW: 224 kDa) is shown. Full-length blots are presented in Supplementary Figure S6. (C) Evaluation of myotube formation of Tet2 knockdown C2C12 cells. Myotubes were visualized by immunostaining for MyHC 6 d after differentiation induction (left). Nuclei were stained with DAPI. Scale bar, 100 μm. The fusion index was calculated as the ratio of the number of nuclei in MyHC-positive cells to the total number of nuclei present in the observation field. The quantitative analysis revealed a significant decrease in the fusion index of shTet2 C2C12 as compared with shCtrl cells (right). Data are presented as means ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: Knockdown, shRNA, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot, Immunostaining, Staining

Journal: Scientific reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro.

doi: 10.1038/srep43539

Figure Lengend Snippet: Figure 4. Promoter methylation status of myoblast differentiation-associated genes in Tet2 knockdown C2C12. C2C12 cells transfected with Tet2 shRNA (shTet2) or control shRNA (shCtrl) were cultured in growth medium. Methylation status of promoters of myogenin (A), Myf6 (B) or myomaker (C) was analyzed by using bisulfite sequencing. Numbers with a minus sign indicate the position of CpG relative to the transcription starting site. Each row represents an individual clone sequenced; black and white circles represent methylated and unmethylated CpGs, respectively. Percentage of methylation indicates the proportion of methylated CpG sites relative to the whole CpG sites examined.

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: Methylation, Knockdown, Transfection, shRNA, Control, Cell Culture, Methylation Sequencing

Journal: Scientific reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro.

doi: 10.1038/srep43539

Figure Lengend Snippet: Figure 5. Knockdown of Tet2 decreases the ability of vitamin C to enhance 5hmC formation and to promote the expression of myoblast differentiation-associated genes. C2C12 cells transfected with Tet2 shRNA (shTet2) or control shRNA (shCtrl) were cultured in growth medium with or without vitamin C (VC; 500 μM). (A) Immunostaining for 5hmC in shTet2 vs shCtrl C2C12 cells with or without VC treatment. Nuclei were stained with DAPI. Scale bar, 50 μm. (B) Quantification of fluorescence intensities of 5hmC. The quantitative analysis revealed that Tet2 knockdown decreased the ability of VC to enhance 5hmC formation. (C) qRT-PCR analysis for the expression of myogenin, Myf6 and myomaker in shTet2 vs shCtrl C2C12 cells with or without VC treatment. Gapdh was used as an internal control. Data are presented as means ± SEM (n = 3). Asterisks above columns represent significant difference among groups (p < 0.05).

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: Knockdown, Expressing, Transfection, shRNA, Control, Cell Culture, Immunostaining, Staining, Fluorescence, Quantitative RT-PCR

Journal: Scientific reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro.

doi: 10.1038/srep43539

Figure Lengend Snippet: Figure 6. Knockdown of Tet2 decreases the ability of vitamin C to promote myoblast differentiation. C2C12 cells transfected with Tet2 shRNA (shTet2) or control shRNA (shCtrl) were subdivided into two experimental groups: non-vitamin C treatment group (indicated as VC−), in which VC was absent in both growth medium and differentiation medium, and VC-treatment group (indicated as VC+), in which the cells were first cultured for 48 h in growth medium containing 500 μM VC, and then were shifted to differentiated medium containing 500 μM VC. (A) Evaluation of myoblast differentiation in different treatment groups. Cells after 6 d of differentiation induction were immunostained with anti-MyHC antibodies to mark the myotubes. Nuclei were stained with DAPI. Scale bar, 100 μm. (B) Quantification analysis for differentiation efficiency in different treatment groups. The fusion index was calculated as the ratio of the number of nuclei in MyHC- positive cells to the total number of nuclei present in the observation field. (C) qRT-PCR analysis for the expression of myogenin, Myf6 and myomaker in C2C12 cells after 4 d of differentiation induction in different treatment groups. Gapdh was used as an internal control. Data are presented as means ± SEM (n = 3). Asterisks above columns represent significant difference among groups (p < 0.05).

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: Knockdown, Transfection, shRNA, Control, Cell Culture, Staining, Quantitative RT-PCR, Expressing

Journal: Theranostics

Article Title: The LRP4/YAP axis drives the radiation-tolerant persister (RTP) cell state in breast cancer

doi: 10.7150/thno.101393

Figure Lengend Snippet: A RNAi screen for the identification of functional regulator of radiation-induced cell plasticity. A . schematic representation of RNAi screening strategy. B-C . Proportion of ALDH br cells in BFP + ( B ) and RFP + SUM159 cells ( C ) following silencing of each individual gene contained in the RNAi library and normalized with non-targeting siRNA (siCTRL). Genes targeted by a siRNA inducing a significant reduction of the ALDH br cell proportion are highlighted. Statistical test used is Student's t-test. Data represent mean ± SD D . Representative images of the high-content screening captures. ALDEFLUOR cellular staining is represented in green. Dead cells are labeled by DRAQ5 in red. RFP+ cells are in yellow and BFP+ cells in blue. E-F . Validation of candidate genes using FACS analysis. Proportion of ALDH br cells in BFP + ( E ) and RFP + SUM159 cells ( F ) are represented normalized with non-targeting siRNA (siCTRL). siRNA targeting ALDH1A1 was used as positive control and siRNA targeting TEXT12 or VDACL were used as negative control. Statistical test used is Student's t-test. Data represent mean ± SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: To evaluate the effect of LRP4 silencing on radiotherapy-response, SUM159, S68, and MDA-MB-231 cells were first transduced with a lentivirus expressing shRNA constructs (shLRP4, #SHCLNV or shCTRL, #SHC201VN (empty vector); Merck Sigma-Aldrich)..Cells shCTRL or shLRP4 were plated at low-density (between 75 and 2000 cells per well, depending on the dose) in 12-well plates (Falcon, 353043), with 3.5mL of culture medium and irradiated between 0 and 6Gy using Synergy linear accelerator (Elekta).

Techniques: Functional Assay, High Content Screening, Staining, Labeling, Biomarker Discovery, Positive Control, Negative Control

Journal: Theranostics

Article Title: The LRP4/YAP axis drives the radiation-tolerant persister (RTP) cell state in breast cancer

doi: 10.7150/thno.101393

Figure Lengend Snippet: LRP4/YAP axis inhibition radiosensitizes iCSC. A-B . Proportion of cells ( A , SUM159; B , S68) presenting a nuclear location of YAP in each cell subpopulations five days post-irradiation, with representative images of YAP staining (in green) in each cell subpopulations on the right panel. Nuclei are counterstained with DAPi (in blue). C . Pre-ranked GSEA interrogating differential expression between iCSC and late non-CSC and YAP/TAZ target genes. D . Western blot of markers related to YAP/TAZ signaling and its activation in SUM159 and S68 cells silenced for LRP4 (shLRP4) compared to the non-targeting shRNA (shCTRL). The mean intensities are indicated below each band for each condition. E . Heat map representing the mRNA expression of the LRP4, ALDH1A1, and YAP/TAZ target genes in SUM159 and S68 silenced for LRP4 (siLRP4) compared to the non-targeting siRNA (siCTRL). Each row represents three independent replicates per conditions (R1, R2, and R3). F . Proportion of SUM159 cells presenting a nuclear location of YAP in late non-CSC and iCSC following LRP4 silencing (siLRP4) compared to a non-targeting siRNA (siCTRL). G-H . SUM159 ( G ) and S68 cells ( H ), following LRP4 silencing (shLRP4) compared to a non-targeting siRNA (shCTRL), were exposed to various dose of radiation therapy and subjected to clonogenic survival assays ( G ), with representative images (right panels). I . Patient-derived xenograft organoid (PDXO) size distribution for CRCM389 cells WT (shCTRL) or silenced for LRP4 (shLRP4) following irradiation (RT) and compared to untreated condition (CTRL) (left panel). Representative pictures of PDXO-CRCM389 7 days post-treatment (right panel). Statistical test used is Student's t-test. Data represent mean ± SD. ns (not significant), *p<0.05, **p<0.01, ***p<0.001. J . Kaplan-Meier tumor-free survival curves of mice xenografted with 100,000-200,000 SUM159 irradiated cells silenced for LRP4 (shLRP4) compared to the control (shCTRL). p-value and hazard ration (HR) estimated according to Log-rank (Mantel-Cox) test.

Article Snippet: To evaluate the effect of LRP4 silencing on radiotherapy-response, SUM159, S68, and MDA-MB-231 cells were first transduced with a lentivirus expressing shRNA constructs (shLRP4, #SHCLNV or shCTRL, #SHC201VN (empty vector); Merck Sigma-Aldrich)..Cells shCTRL or shLRP4 were plated at low-density (between 75 and 2000 cells per well, depending on the dose) in 12-well plates (Falcon, 353043), with 3.5mL of culture medium and irradiated between 0 and 6Gy using Synergy linear accelerator (Elekta).

Techniques: Inhibition, Irradiation, Staining, Quantitative Proteomics, Western Blot, Activation Assay, shRNA, Expressing, Derivative Assay, Control

Journal: Theranostics

Article Title: WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

doi: 10.7150/thno.25025

Figure Lengend Snippet: WNT6 is overexpressed at the mRNA and protein levels in GBM. (A) Expression levels of WNT6 in 27 lower-grade gliomas (LGG; grey dots), 572 glioblastomas (GBM, WHO grade IV; black and colored dots) and 10 unmatched normal brains (black unfilled dots) from TCGA. GBM molecular subtypes are represented as colors (red = classical; blue = proneural; green = neural; yellow = mesenchymal). WNT6 is overexpressed (TCGA data level 3 values ≥ 0.41; above red dashed line) in 16% (n = 89) of GBM samples. (B) WNT6 protein expression in WHO grades I-IV glioma samples from Hospital Santo António cohort assessed by IHC (n = 63; p = 0.037; chi-squared test). Representative images are shown for a WNT6-negative GBM (a), high WNT6 expression in GBM (b) and WHO grade III anaplastic oligoastrocytoma (c), and a WHO grade II diffuse oligodendroglioma with intermediate levels of WNT6 expression (d). WNT6 staining was mostly cytoplasmic in glioma cells (closed arrowheads) and not present in lymphocytes (e; arrow), being almost exclusively negative for endothelial cells (b-d and f; open arrowheads). Bottom graph summarizes IHC data for the whole dataset. LGG: lower-grade glioma; TCGA: The Cancer Genome Atlas; WHO: World Health Organization.

Article Snippet: Cells were transfected with WNT6 gene-specific shRNA expression vector inserted in a pRS plasmid (TR308360, clones TI333434 and TI333435 from Origene; named U373-shWNT6 or SNB19-shWNT6, respectively) or with a scrambled negative control non-effective shRNA in a pRS plasmid (TR30012, Origene; named U373-shCtrl or SNB19-shCtrl).

Techniques: Expressing, Staining

Journal: Theranostics

Article Title: WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

doi: 10.7150/thno.25025

Figure Lengend Snippet: WNT6 promotes GBM aggressiveness in vitro . (A-B) The efficiency of WNT6 silencing in U373 and SNB19 glioblastoma cells was analyzed by qRT-PCR and WB (A), and immunofluorescence (B). (A) Top: WNT6 expression levels were normalized to TBP . Bottom: WB images are representative of 3 independent assays; α-tubulin was used as reference protein. (B) DAPI was used to stain the nucleus (40x magnification; scale bar = 50 µm). (C-D) Cell viability was measured by trypan blue (C) and MTT (D) assays in shCtrl and shWNT6 cells. (E) Cell proliferation was evaluated by BrdU incorporation. (F-G) Matrigel invasion assays were used to assess the cells' invasion capacity. Representative images (F) with cell nuclei stained with DAPI (scale bar = 250 µm) and quantification (G) of invasive U373 and SNB19 shCtrl and shWNT6 cells. (H-K) Migration (wound healing) assays were performed in U373 (H-I) and SNB19 (J-K) shCtrl and shWNT6 cells to assess the cells' migration capacity. Representative micrographs throughout time for U373 (H) and SNB19 (J) cells (40x magnification; scale bar = 250 µm). The wound gap was measured and expressed as a percentage of wound closure for U373 (I) and SNB19 (K) cells (**** p <0.001; two-way ANOVA post-hoc Sidak's test). Results represent data from at least 3 independent experiments (mean ± SD). *, p < 0.05; **, p < 0.01; ***, p < 0.005 and ****, p < 0.001 (unless otherwise stated, a two-sided unpaired t -test with Welch's correction was applied when homoscedasticity was not verified).

Article Snippet: Cells were transfected with WNT6 gene-specific shRNA expression vector inserted in a pRS plasmid (TR308360, clones TI333434 and TI333435 from Origene; named U373-shWNT6 or SNB19-shWNT6, respectively) or with a scrambled negative control non-effective shRNA in a pRS plasmid (TR30012, Origene; named U373-shCtrl or SNB19-shCtrl).

Techniques: In Vitro, Quantitative RT-PCR, Immunofluorescence, Expressing, Staining, BrdU Incorporation Assay, Migration

Journal: Theranostics

Article Title: WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

doi: 10.7150/thno.25025

Figure Lengend Snippet: WNT6 promotes resistance to temozolomide chemotherapy in GBM cells. (A-B) U373 (A) and SNB19 (B) shCtrl- and shWNT6-transfected cells were treated with vehicle or TMZ for 6 days, and cell death was measured by Annexin-V and PI. The graphs present data of 3 independent assays (mean ± SD). Representative dot plots are displayed below. *, p < 0.05; **, p < 0.01; ***, p < 0.001 and ****, p < 0.0001 (two-way ANOVA post-hoc Tukey's test). DMSO: dimethyl sulfoxide; PI: propidium iodide; TMZ: temozolomide.

Article Snippet: Cells were transfected with WNT6 gene-specific shRNA expression vector inserted in a pRS plasmid (TR308360, clones TI333434 and TI333435 from Origene; named U373-shWNT6 or SNB19-shWNT6, respectively) or with a scrambled negative control non-effective shRNA in a pRS plasmid (TR30012, Origene; named U373-shCtrl or SNB19-shCtrl).

Techniques: Transfection

Journal: Theranostics

Article Title: WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

doi: 10.7150/thno.25025

Figure Lengend Snippet: WNT6 silencing decreases GBM stem cell features. (A) SOX2 and NESTIN double-immunostaining in U373 (left) and SNB19 (right) shCtrl/shWNT6 cells. DAPI was used to stain cell nuclei (40x magnification; scale bar = 50 µm). (B) Representative images of U373 (left) and SNB19 (right) shCtrl/shWNT6 neurospheres at days 10 and 15 after plating, respectively (40x magnification; scale bar = 250 µm). (C) Quantification of neurospheres formation (n = 3 independent assays; mean ± SD; *, p < 0.05; **, p < 0.01, two-sided unpaired t -test with Welch's correction being applied when homoscedasticity was not verified). (D) Limiting dilution assays in U373-shCtrl (black) and -shWNT6 (red) cells to assess their sphere-forming capacity. The graph is representative of 3 independent assays with similar results. The trend lines represent the estimated active cell frequency ( p = 0.01, likelihood ratio test). (E) Representative images of U373 (left) and SNB19 (right) shCtrl/shWNT6 cells cultured under stem cell-adherent conditions (scale bar = 250 µm). (F) MTT assay evaluating U373 (left) and SNB19 (right) shCtrl/shWNT6 cell viability when cultured under stem cell-adherent conditions (n = 3 independent assays; mean ± SD; **, p < 0.01; ****, p < 0.0001; two-sided unpaired t -test with Welch's correction applied when homoscedasticity was not verified). (G) Heatmap representation of expression levels of WNT6 and 30 stem cell-related genes in 573 GBM patients' samples from TCGA. Each column corresponds to a patient and each line to a gene. Expression values increase from darker blue (low levels) to darker red (high levels). (H) Correlation graphs between WNT6 expression (x-axis) and the expression of stem cell genes selected based on the heatmap (y-axis; Pearson's correlation test; r and p values are indicated).

Article Snippet: Cells were transfected with WNT6 gene-specific shRNA expression vector inserted in a pRS plasmid (TR308360, clones TI333434 and TI333435 from Origene; named U373-shWNT6 or SNB19-shWNT6, respectively) or with a scrambled negative control non-effective shRNA in a pRS plasmid (TR30012, Origene; named U373-shCtrl or SNB19-shCtrl).

Techniques: Double Immunostaining, Staining, Cell Culture, MTT Assay, Expressing, Gene Expression

Journal: Theranostics

Article Title: WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

doi: 10.7150/thno.25025

Figure Lengend Snippet: WNT6 accelerates GBM-associated death in an intracranial mouse model. U373 transfected cells were orthotopically injected in the brain of NSG mice (n = 8 per group). (A) Weight curves after tumor implantation. (B) Kaplan-Meier overall survival curves of mice ( p = 0.0042; Log-rank test). (C-G) Post-mortem brain histological and molecular analyses. (C-D) H&E and anti-WNT6 IHC staining were used as controls for GBM formation and successful long-term WNT6 silencing, respectively. (E-G) IHC for cell proliferation/viability markers Ki-67 and Cyclin D1 (E), stem cell markers SOX2 and NESTIN (F), and anti-apoptotic marker BCL2 (G). Scale bars' values are shown in each microphotograph (20 µm or 50 µm). H&E: hematoxylin and eosin.

Article Snippet: Cells were transfected with WNT6 gene-specific shRNA expression vector inserted in a pRS plasmid (TR308360, clones TI333434 and TI333435 from Origene; named U373-shWNT6 or SNB19-shWNT6, respectively) or with a scrambled negative control non-effective shRNA in a pRS plasmid (TR30012, Origene; named U373-shCtrl or SNB19-shCtrl).

Techniques: Transfection, Injection, Tumor Implantation, Immunohistochemistry, Marker

Journal: Theranostics

Article Title: WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

doi: 10.7150/thno.25025

Figure Lengend Snippet: WNT6 influences the activation status of WNT, SFK, and STAT pathways in GBM. Phospho-kinase arrays were performed to evaluate the phosphorylation status of cancer-related kinases in U373 (A) and SNB19 (B). Each array includes 2 technical replicates; 2 and 1 biological replicates were used for U373 and SNB19, respectively. Fold changes between phosphorylation levels of proteins in shWNT6 vs. shCtrl cells are shown (mean ± SD). HSP60 was used as reference protein. Similar alterations in the phosphorylation of particular proteins for U373 and SNB19 cells are highlighted in red. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.0001; two-sided unpaired t -test. (C-D) The phosphorylation status of STAT3, β-catenin, and AKT, and their respective total forms were assessed in U373 (C) and SNB19 (D) shCtrl and shWNT6 cells by WB (images are representative of 3 independent assays; α-tubulin was used as reference protein for normalization; relative expression levels are shown above each band). (E) GSEA analyses identifying signatures of genes significantly associated (ES > 0.3, and FDR < 0.3) with WNT6 in GBM patients from TCGA. ES: enrichment score; FDR: false discovery rate.

Article Snippet: Cells were transfected with WNT6 gene-specific shRNA expression vector inserted in a pRS plasmid (TR308360, clones TI333434 and TI333435 from Origene; named U373-shWNT6 or SNB19-shWNT6, respectively) or with a scrambled negative control non-effective shRNA in a pRS plasmid (TR30012, Origene; named U373-shCtrl or SNB19-shCtrl).

Techniques: Activation Assay, Phospho-proteomics, Expressing

Journal: Theranostics

Article Title: WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

doi: 10.7150/thno.25025

Figure Lengend Snippet: WNT6 expression is prognostically valuable in GBM patients. Kaplan-Meier survival curves of WNT6 -low and WNT6 -high GBM patients derived from qRT-PCR data (A) or microarray (B-E) data. (A) Portuguese dataset, n = 51, median OS 16.9 vs. 10.4 months (low vs. high WNT6 expression, respectively; p = 0.032, Log-rank test). (B) Freije dataset, n = 59, median OS 9.6 vs. 5 months ( p = 0.006, Log-rank test). (C) Gravendeel dataset, n = 159, median OS 10.1 vs. 6 months ( p = 0.029, Log-rank test). (D) Vital dataset, n = 26, median OS 18 vs. 4 months ( p = 0.02, Log-rank test). (E) Forest plot of hazard ratios (HR), illustrating the associations between WNT6 expression and overall survival in GBM patients. Each cohort is accompanied by its HR (black square; the size of the square represents the weight of the study in the meta-analysis) and 95% confidence intervals (extending lines). The diamond represents the estimated pooled effect (HR = 1.637; 95% confidence interval: 1.285 - 2.086; p < 0.0001). CI: confidence interval; HR: hazard ratio.

Article Snippet: Cells were transfected with WNT6 gene-specific shRNA expression vector inserted in a pRS plasmid (TR308360, clones TI333434 and TI333435 from Origene; named U373-shWNT6 or SNB19-shWNT6, respectively) or with a scrambled negative control non-effective shRNA in a pRS plasmid (TR30012, Origene; named U373-shCtrl or SNB19-shCtrl).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Microarray

Journal: Theranostics

Article Title: WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

doi: 10.7150/thno.25025

Figure Lengend Snippet: High levels of WNT6 expression are significantly associated with shorter survival of GBM patients (n = 407; microarray data).

Article Snippet: Cells were transfected with WNT6 gene-specific shRNA expression vector inserted in a pRS plasmid (TR308360, clones TI333434 and TI333435 from Origene; named U373-shWNT6 or SNB19-shWNT6, respectively) or with a scrambled negative control non-effective shRNA in a pRS plasmid (TR30012, Origene; named U373-shCtrl or SNB19-shCtrl).

Techniques: Expressing, Microarray

Journal: Theranostics

Article Title: WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma

doi: 10.7150/thno.25025

Figure Lengend Snippet: Proposed model for the functional, molecular and clinical impact of WNT6 in GBM. WNT6-high GBM cells present increased proliferation, invasion, migration, TMZ resistance and stem cell self-renewal capacities, which are accompanied by activation of the WNT, SFK, RTK, STAT and AKT pathways. Clinically, WNT6-high tumors cause shorter overall survival (both in mouse GBM models and human patients).

Article Snippet: Cells were transfected with WNT6 gene-specific shRNA expression vector inserted in a pRS plasmid (TR308360, clones TI333434 and TI333435 from Origene; named U373-shWNT6 or SNB19-shWNT6, respectively) or with a scrambled negative control non-effective shRNA in a pRS plasmid (TR30012, Origene; named U373-shCtrl or SNB19-shCtrl).

Techniques: Functional Assay, Migration, Activation Assay