Journal: Advanced Science

Article Title: Delivery of A Chemically Modified Noncoding RNA Domain Improves Dystrophic Myotube Function

doi: 10.1002/advs.202410908

Figure Lengend Snippet: Optimization of RNA‐based CYTOR exon 2 delivery. ​​A) Normalized CYTOR expression at different time points in HEK293 cells after modified and unmodified CYTOR exon 2 RNA or DNA plasmid delivery. N = 4. B–E) Time course of normalized gene expression of genes related to the innate immune response ( TNFα , IL‐6 ) and genes induced by pattern recognition receptor activation ( NFκB , IFNα ) upon treatment with modified and unmodified CYTOR exon 2 RNA or DNA plasmid delivery. N = 4. F) CYTOR RNA abundance from (A) at the 24 h time point. N = 4. * p < 0.05, ** p < 0.01.

Article Snippet: In short, Anti‐Reverse Cap Analog (ARCA, NEB #S1411) using T7 RNA Polymerase was used for the capped CYTOR exon 2 RNA by combining the ARCA/NTP mix, T7 RNA Polymerase Mix and a suitable DNA template (NEB, E2060S).

Techniques: Expressing, Modification, Plasmid Preparation, Gene Expression, Activation Assay

Journal: Advanced Science

Article Title: Delivery of A Chemically Modified Noncoding RNA Domain Improves Dystrophic Myotube Function

doi: 10.1002/advs.202410908

Figure Lengend Snippet: Forced expression of human CYTOR exon 2 in mouse‐induced myogenic progenitor and embryonic stem cells. A) Normalized gene expression in mouse embryonic stem cells exogenously expressing a GFP control construct or CYTOR exon2 from a DNA vector. N = 5‐6. B) Normalized gene expression in mouse embryonic stem cells after transfection of chemically modified CYTOR exon2 RNA. N = 5‐6. C) Normalized gene expression in induced myogenic progenitors expressing a GFP control construct or CYTOR exon2 from a DNA vector. N = 3. D) Normalized gene expression in induced myogenic progenitors after transfection of CYTOR exon2,ΨU RNA. N = 3. * p < 0.05, ** p < 0.01.

Article Snippet: In short, Anti‐Reverse Cap Analog (ARCA, NEB #S1411) using T7 RNA Polymerase was used for the capped CYTOR exon 2 RNA by combining the ARCA/NTP mix, T7 RNA Polymerase Mix and a suitable DNA template (NEB, E2060S).

Techniques: Expressing, Gene Expression, Control, Construct, Plasmid Preparation, Transfection, Modification

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: An image of a haematoxylin and eosin (HE)-stained section of HT1080 tumours showing the presence of abundant vascular channels containing red blood cells is illustrated. A magnified image of these vascular channels is depicted on the right hand side. (Original magnifications 100X and 400X respectively) B. Paraffin sections of HT1080 tumours were subjected to immuno histochemistry (IHC) analyses using antibodies to various angiogenic markers. The tumour cells were positive for PECAM, VE-Cadherin, VEGF, NRP-1, VEGF 165 and VEGFR-2 (violet colour). PECAM positive micro-vessel formation is indicated by a black arrow (top layer, right panel). C. Nuclear localization of HIF-1α in the tumour cells confirms the presence of in situ hypoxia. The sections were counterstained with hematoxylin to demarcate the nuclei (blue).

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Staining, Immunohistochemistry, In Situ

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: Confocal microscopy analyses show that the hypoxic cells exhibit up-regulation of NRP-1 ( A ) and nuclear stabilization of HIF-1α ( B ). Nuclei are demarcated by DAPI (Blue). Mean fluorescence intensity (M.F.I.) of the cells was measured by Image J software (NIH) at membrane (for NRP-1) and in the nuclear region (for HIF-1α). The M.F.I. of 30 randomly selected cells was used to calculate mean ± S.E.M. The analyses have been graphically depicted (b and d for NRP-1 and HIF-1α respectively) ** p<0.01 and *** p<0.001. C. Quantitative PCR analyses for NRP-1 and HIF-1α mRNA show 2.4 and 1.7 folds up-regulation of these genes in the cells incubated under hypoxia compared to normoxia. (N = 3; *p<.05 and ** p<.01). D. Western blot experiments performed on the cells grown under normoxia vs. hypoxia show that the protein levels of both HIF-1α (upper panel) and NRP-1 (middle panel) are up-regulated in the hypoxic cells compared to the normoxic ones (∼1.4 and 3.2 folds respectively). E. Results of quantitative PCR experiments showed that the hypoxia-induced up-regulation of NRP-1 and VEFG 165 mRNA was sensitive to the presence of chetomin in the medium, indicating that these genes are down-stream events in the HIF-1α-mediated transcription process. F. Confocal microscopy analysis shows that the hypoxia-mediated up-regulation of NRP-1 protein (Cyan, upper panel) is abrogated in the presence of chetomin in the medium (lower panel), suggesting that NRP-1 expression critically depends on the HIF-1α -mediated transcription. Nuclei are demarcated by DAPI (Blue).

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Confocal Microscopy, Fluorescence, Software, Real-time Polymerase Chain Reaction, Incubation, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: Silencing of NRP-1 expression in the HT/shNRP-1 cells was validated at transcript level by performing quantitative PCR experiments. The NRP-1 mRNA was ∼500 folds down-regulated in the shNRP-1 clone (N = 3; ***p<0.001) showing that the shRNA constructs used were effective. B. The results obtained in the PCR experiments were validated by western blot experiments. The NRP-1 expression was down-regulated in the HT/shNRP-1 cells at protein level as well. C. Quantitative PCR analyses confirmed the higher expression of NRP-1 transcript in the HT/flNRP-1 clone compared to the HT1080/Scr cells (∼2 folds, N = 3; *p<0.05). D. Western blot experiments were performed on the HT1080/Scr, HT/shNRP-1 and HT/flNRP-1 cells. Data show the presence of a 150 kDa band of the NRP-GFP fusion protein in the HT/flNRP-1 cells when the blot was probed with antibodies to NRP-1 (upper panel) and GFP (middle panel). The down-regulation of NRP-1 in the HT/shNRP-1 clone is also seen (upper panel). E. Confocal microscopy analyses show a higher expression of NRP-1 in the HT/flNRP-1 clone (upper panel) and a reduced expression of NRP-1 in HT/shNRP-1 clone (Lower panel) compared to the HT1080/Scr cells (middle panel). Membrane-localized NRP-1-GFP fusion protein is seen in the HT/flNRP-1 cells. The RFP fluorescence is seen in the nuclei of HT1080/Scr and HT/shNRP-1 cells respectively. Nuclei are demarcated by DAPI (Blue).

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, shRNA, Construct, Western Blot, Confocal Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: Expression of PECAM and VEGFR-2 mRNA was quantified by performing real time PCR experiments on the HT1080/WT cells that were incubated under normoxia and hypoxia – with or without chetomin. Both PECAM and VEGFR-2 mRNAs were up-regulated by hypoxia. Presence of chetomin abrogated the up-regulation of PECAM and VEGFR2 genes by hypoxia indicating that these genes are regulated by the HIF-1α-mediated transcription. B. Quantitative PCR experiments performed on the HT1080/Scr and HT/shNRP-1 cells incubated under hypoxia show that hypoxia failed to up-regulate the expression of PECAM, VEGF 165 as well as VEGFR-2 in the HT/shNRP-1 cells, indicating that the hypoxia-induced angiogenic program in the HT1080 cells is controlled by NRP-1. C. Tubule formation on matrigel by HT1080/Scr and HT/shNRP-1 cells under normoxia and hypoxia is depicted. The HT/shNRP-1 cells failed to form tubules even after priming with hypoxia (lower right hand panel), indicating that NRP-1 expression in critical for the enhanced tubule formation by the hypoxia-primed HT1080 cells. The hypoxia-primed wild type cells formed robust tubules as seen in the earlier experiments (upper right hand panel). D. HT/flNRP-1 cells form dense tubules even without the hypoxia-priming. The tubule formation by these cells started very early (2 hours) and became very dense by 6 hours (upper right hand panel). After 6 hours, the tubules collapsed as the HT/flNRP-1 cells invaded the matrigel vigorously (lower panel) forming a monolayer in the well. NRP-1 controls the tumorigenic properties of HT1080 cells. E. Matrigel-invasion property of HT1080/Scr cells was compared with that of HT/flNRP-1 and HT/shNRP-1 cells. A representative image of the invaded cells stained with crystal violet is depicted (Original magnification: 100X). Quantification of the invaded cells showed that the HT/flNRP-1 cells possessed significantly enhanced invasive property (N = 3; ** p<0.01) while the HT/shNRP-1 cells showed a significantly reduced invasive ability (N = 3; ***p<0.001). F. Anchorage-independent growth of HT1080/Scr, HT/shNRP-1 and HT/flNRP-1 was examined by performing soft agar colony assay. A representative phase contrast image of the colonies formed by these cells is illustrated (original magnification: 100X). The HT/flNRP-1 cells formed large colonies having loose migrating cells at the border (middle panel), while the HT/shNRP-1 cells formed very small compact colonies. Quantification of the colony formation shows that the number of colonies formed by the HT/flNRP-1 cells was significantly higher (**p<0.01) while that by the HT/shNRP-1 cells was significantly lower (* p<0.05) compared to the HT1080/Scr cells. Data show that NRP-1 expression is necessary for the anchorage-independent growth of HT1080 cells. Data are represented as mean ± S.E.M.

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Staining, Colony Assay

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: HT1080/Scr, HT/flNRP-1 and HT/shNRP-1 cells were injected subcutaneously in the flanks of NOD/SCID mice (9 mice per set) to compare their tumorgenic potential. HT/flNRP-1 cells formed larger tumours (mouse in the middle) compared to those formed by the HT1080/Scr cells (mouse on the left). HT/shNRP-1 cells did not form tumour (mouse on right, 0/9). B. A comparative image of the tumours formed by the HT1080/Scr and the HT/flNRP-1 cells is depicted. The tumours formed by HT1080/Scr and HT/flNRP-1 cells were weighed. The mean weight of the tumours formed by the HT/flNRP-1 was significantly higher than that of the HT1080/Scr cells. (N = 9, ***p<0.001) C. The kinetics of tumour formation by the HT/flNRP-1 cells was significantly higher compared to that of HT1080/Scr cells (**p<0.01, ***p<0.001). D. HT/flNRP-1 tumor sections were subjected to IHC analyses using antibodies to various angiogenic markers like PECAM, VE-Cadherin, VEGF, VEGF 165 , NRP-1 and VEGFR-2. The sections showed a strong expression of all these markers indicating that the HT/flNRP-1 tumours are highly angiogenic (original magnification: 200X). E. Paraffin sections of the tumours formed by the HT1080/Scr and the HT/flNRP-1 were immuno-stained with an anti-PECAM antibody. The HT/flNRP-1 tumours showed a high density of PECAM-positive tube-like structures (indicated by arrows) compared to the HT1080/Scr tumours indicating a high level of angiogenesis (original magnification: 100X) F. The strong nuclear localization of HIF-1α seen in these tumours confirms a high level of in situ hypoxia (original magnification: 200X). Hypoxia-priming enhances the in vivo tumour formation. G. HT1080/Scr and HT/flNRP-1 cells – primed or not with hypoxia – were injected sub-cutaneously in the skin of NOD/SCID mice. The size of the tumours formed by the hypoxia-primed cells was significantly larger that their respective normoxic controls. Quantification of the tumour weights (N = 9) shows that hypoxia-priming significantly enhances the tumour formation by both HT1080/Scr as well as by the HT/flNRP-1 cells (Data are represented as mean± S.E.M., N = 9, ***p<.001 and **p<.01.) H. The kinetics of tumour formation by the hypoxia-primed cells at various time points was significantly higher compared to their normoxic counterparts (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Injection, Expressing, Staining, In Situ, In Vivo

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: Quantitative PCR analyses for OCT3/4, c-Myc and KLF4 mRNA shows that hypoxia up-regulated the expression of OCT3/4 and c-Myc while down-regulates that of KLF4. B. Presence of chetomin in the incubation medium of cells incubated under hypoxia resulted in a further up-regulation of the expression of OCT3/4 and c-Myc mRNA indicting that these genes are independent of HIF-1α-mediated transcription and is under the control of a mechanism that is negatively regulated by the HIF-1α-mediated transcription. The down-regulation of KLF4 by hypoxia was however, rescued by chetomin showing that it was a HIF1-α-dependent process. C. Quantification of OCT3/4, c-Myc and KLF4 mRNA in the hypoxia-primed HT1080/Scr and HT/shNRP-1 cells shows that the expression of OCT3/4 and c-Myc is significantly higher in the hypoxia-primed HT/shNRP-1 cells compared to the hypoxia-primed HT1080/Scr cells, showing that the up-regulation of these genes by hypoxia is not only NRP-1 independent, but also gets further enhanced when NRP-1 is silenced.The suppression of KLF4 expression by hypoxia was partially rescued by silencing of NRP-1 indicating the role of HIF-1α-NRP-1 axis in its down-regulation by hypoxia. (Data in all panels are represented as mean ± S.E.M; N = 3 and *** p<0.001, ** p<0.01 and *p<0.5).

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Incubation

Journal: Virology

Article Title: HPV16-transformed human keratinocytes depend on SIX1 expression for proliferation and HPV E6/E7 gene expression

doi: 10.1016/j.virol.2019.08.009

Figure Lengend Snippet: Real-time RT/PCR with E6- and E7-specific primers, and SIX1 primers, on RNA isolated from mock-transfected HKc/DRd-1 (D1DR) and HKc/DRd-1 transiently-transfected, in triplicate, with two different siRNA’s against HPV16 E7. RNA was isolated 48 h after transfection. Data are normalized against GAPDH and are expressed relative to the values obtained for mock-transfected HKc/DRd-1. Error bars represent SD.

Article Snippet: Retroviral Infection PA317 cells were transfected with four different pSuper-shRNA constructs (Origene, Rockville, MD) against SIX1, targeting four different positions within the human SIX1 mRNA sequence.

Techniques: Quantitative RT-PCR, Isolation, Transfection

Journal: Virology

Article Title: HPV16-transformed human keratinocytes depend on SIX1 expression for proliferation and HPV E6/E7 gene expression

doi: 10.1016/j.virol.2019.08.009

Figure Lengend Snippet: The pre-malignant, HPV16-immortalized, differentiation-resistant HKcDR-d1 cell line at 70% confluency was transfected with pSuper vectors containing either a specific shRNA against human SIX1 (SIX1-sh, at 4 or 5 mg/well in six-well plates, three wells for each dose) or a scrambled, control shRNA (SCRAM-sh, 5 mg/well in six-well plates). A, RT PCR results for SIX1; B, proliferation curves for the cell lines resulting from the transfection: 5,000 cells were plated per well in 24-well plates (1 ml medium/well). Cells in triplicate wells were trypsinized and counted at the times indicated over a 7-day period using a hemocytometer. Bars represent standard deviations.

Article Snippet: Retroviral Infection PA317 cells were transfected with four different pSuper-shRNA constructs (Origene, Rockville, MD) against SIX1, targeting four different positions within the human SIX1 mRNA sequence.

Techniques: Transfection, shRNA, Reverse Transcription Polymerase Chain Reaction

Journal: Virology

Article Title: HPV16-transformed human keratinocytes depend on SIX1 expression for proliferation and HPV E6/E7 gene expression

doi: 10.1016/j.virol.2019.08.009

Figure Lengend Snippet: A, unsupervised hierarchical clustering of differentially expressed genes between HKc/DR control siRNA, and HKc/DR SIX1-siR2. The color represents the expression level of a gene above (red), below (green), or at neutral (black) the mean expression level of that gene across all samples. B, heatmap of selected genes relevant to MET in the SIX1- siR2 dataset. C, canonical pathways and diseases & functions differentially affected by SIX1 loss in HKc/DRd-1 identified by Ingenuity Pathway Analysis (IPA) of the SIX1-siR2 dataset (1.4 fold change up and down; p-value < 0.01).

Article Snippet: Retroviral Infection PA317 cells were transfected with four different pSuper-shRNA constructs (Origene, Rockville, MD) against SIX1, targeting four different positions within the human SIX1 mRNA sequence.

Techniques: Expressing

Journal: Virology

Article Title: HPV16-transformed human keratinocytes depend on SIX1 expression for proliferation and HPV E6/E7 gene expression

doi: 10.1016/j.virol.2019.08.009

Figure Lengend Snippet: Representative fluorescent (A,C,E,G) and phase-contrast (B,D,F,H) images of HKc/DRd-1 transfected with either the pSuper-SCRAM-sh or the pSuper-SIX1sh RNA, co-transfected with pSuper-GFP as described in Materials and Methods. In these experiments, cells were transfected, incubated for the indicated times and photographed, without passaging or antibiotic selection. A,B: control pSuper-SCRAM-sh, 2 days after transfection; C,D: pSuper-SIX1-sh RNA, 2 days after transfection. E,F: control pSuper-SCRAM-sh, 4 days after transfection; G,H: pSuper-SIX1-sh RNA, 4 days after transfection.

Article Snippet: Retroviral Infection PA317 cells were transfected with four different pSuper-shRNA constructs (Origene, Rockville, MD) against SIX1, targeting four different positions within the human SIX1 mRNA sequence.

Techniques: Transfection, Incubation, Passaging, Selection

Journal: Virology

Article Title: HPV16-transformed human keratinocytes depend on SIX1 expression for proliferation and HPV E6/E7 gene expression

doi: 10.1016/j.virol.2019.08.009

Figure Lengend Snippet: A: HKc/DRd-1 were cultured in six-well plates until 60% confluent and transfected in triplicate wells per each siRNA, with two different siRNA constructs against SIX1 as described in Materials and Methods. At the indicated times after transfection, RNA was extracted from the transfected cells, and from parallel cultures of normal human keratinocytes (HKc) and mock-transfected HKc/DRd-1, both utilized as controls. Relative SIX1 and E6/E7 mRNA levels were determined by real-time RT/PCR with primers specific for SIX1, HPV16 E6 and HPV16 E7. Results are averages + standard deviation of triplicate determinations. There was barely detectable SIX1 expression, and no HPV16 E6/E7 expression in normal HKc, which were used here as negative controls. Data are normalized to HKc/DR. B: Correlation of E6/E7 mRNA levels with SIX1 levels (measured by real-time RT/PCR and expressed as % of control) across different experiments with the indicated experimental approaches and tools.

Article Snippet: Retroviral Infection PA317 cells were transfected with four different pSuper-shRNA constructs (Origene, Rockville, MD) against SIX1, targeting four different positions within the human SIX1 mRNA sequence.

Techniques: Cell Culture, Transfection, Construct, Quantitative RT-PCR, Standard Deviation, Expressing

Journal: Virology

Article Title: HPV16-transformed human keratinocytes depend on SIX1 expression for proliferation and HPV E6/E7 gene expression

doi: 10.1016/j.virol.2019.08.009

Figure Lengend Snippet: HKc/DRd-1 were cotransfected with the pLXSN-E7 construct and the pSuper-puro vector, selected with puromycin, and the resulting cell line was utilized for RNA and protein extraction together with control HKc/DRd-1 and their parental line, HKc/HPV16d-1. Real-time RT/PCR was performed in triplicate samples, with primers targeting the HPV16 E6 and E7 sequences and SIX1, and GAPDH as a control for normalization. Data are expressed as % of the GAPDH-normalized levels detected in HKc/DRd-1, for each primer set. Error bars represent SD.

Article Snippet: Retroviral Infection PA317 cells were transfected with four different pSuper-shRNA constructs (Origene, Rockville, MD) against SIX1, targeting four different positions within the human SIX1 mRNA sequence.

Techniques: Construct, Plasmid Preparation, Protein Extraction, Quantitative RT-PCR

Journal: Virology

Article Title: HPV16-transformed human keratinocytes depend on SIX1 expression for proliferation and HPV E6/E7 gene expression

doi: 10.1016/j.virol.2019.08.009

Figure Lengend Snippet: mRNA levels of: A, KRT15 (K15), CTNNβ1, OCLN, VIM, PPARy, PTGS2, and B, EGFR, CTGF, ETS1, MMP1/9 were determined by Realtime RT/PCR analysis in HKc/DR control siR and HKc/DR SIX1-siR2. Data were normalized to GAPDH expression. Bars indicate SD among multiple samples derived from independent transfections (4 for anti-SIX1 siRNA, 3 for control siRNA) as detailed in Materials and Methods.

Article Snippet: Retroviral Infection PA317 cells were transfected with four different pSuper-shRNA constructs (Origene, Rockville, MD) against SIX1, targeting four different positions within the human SIX1 mRNA sequence.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Transfection

Journal: Virology

Article Title: HPV16-transformed human keratinocytes depend on SIX1 expression for proliferation and HPV E6/E7 gene expression

doi: 10.1016/j.virol.2019.08.009

Figure Lengend Snippet: mRNA levels of TGFβRI and TGFβRII were determined by Real-time RT PCR in HKc/DRd-1 control siR and HKc/DRd-1 SIX1-siR2, in comparison with normal HKc (NHKc) and HKc/HPV16. Data were normalized to GAPDH expression. Bars indicate SD among multiple samples derived from independent transfections (4 for anti-SIX1 siRNA, 3 for control siRNA) as detailed in Materials and Methods.

Article Snippet: Retroviral Infection PA317 cells were transfected with four different pSuper-shRNA constructs (Origene, Rockville, MD) against SIX1, targeting four different positions within the human SIX1 mRNA sequence.

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

Journal: Oncogene

Article Title: INCREASED VASCULARITY AND SPONTANEOUS METASTASIS OF BREAST CANCER BY HEDGEHOG SIGNALING MEDIATED UPREGULATION OF CYR61

doi: 10.1038/onc.2011.496

Figure Lengend Snippet: A) CYR61 is notably more abundant in SHH expressing cell secretome compared to vector. Equal numbers of cells from each experimental group were seeded and allowed to attach overnight. The following day cells were washed and cultured in SFM. 18 hrs later, conditioned media was harvested and equal amounts of media were analyzed via western blot for the expression of CYR61. Immuno detection confirmed what was seen in the proteomics analysis. B) Analysis of approximately 1 kB upstream of the transcriptional start site determined that the CYR61 promoter region contains a putative GLI1-recognition sequence that differs from the consensus GLI1-binding region by two nucleotides (underscored) sequence. C) The CYR61 promoter is activated (^p < 0.0001) in breast cancer cell lines expressing SHH. Reporter assays were done using a luciferase-expressing CYR61 promoter in vector-control and SHH-expressing MDA-MB-231 cells. D) ChIP assay confirms that GLI1 binds to the promoter of CYR61. Input: Total native chromatin isolated from cancer cells (indicated, vector-transfected or SHH-expressing); ChIP: PCR amplified DNA product is generated after immunoprecipitation with GLI1 antibody; Water: PCR water-control used to assess quality and lack of contamination; IgG: Isotype control; −ve and +ve control denote kit-provided negative and positive control primers respectively; GLI1-specific primers were used to amplify the putative GLI1 binding site in CYR61 promoter region, and non-specific primers were used as a control to amplify a region 1KB downstream of the putative GLI1 binding in CYR61 promoter that is void of the GLI1 consensus sequence. E) COS7 cells were transfected with CYR61 promoter and empty vector or GLI1-expressing construct. While the wild-type CYR61 promoter bearing the GLI-binding site is significantly upregulated (8-fold; ^p < 0.0001), mutation or deletion of the GLI binding site in the CYR61 promoter results in abrogation (^^p < 0.005) of luciferase reporter activity of the CYR61 promoter indicating that GLI1 transcriptionally regulates CYR61 gene expression.

Article Snippet: SHH and CYR61 mRNA expression in human breast cancer patient tissues was analyzed using tissue qPCR array cDNA samples from OriGene Technologies (Rockville, MD). qPCR was performed per the manufacturers’ recommendations.

Techniques: Expressing, Plasmid Preparation, Cell Culture, Western Blot, Sequencing, Binding Assay, Luciferase, Isolation, Transfection, Amplification, Generated, Immunoprecipitation, Positive Control, Construct, Mutagenesis, Activity Assay

Journal: Oncogene

Article Title: INCREASED VASCULARITY AND SPONTANEOUS METASTASIS OF BREAST CANCER BY HEDGEHOG SIGNALING MEDIATED UPREGULATION OF CYR61

doi: 10.1038/onc.2011.496

Figure Lengend Snippet: A) Stable silencing of endogenous CYR61 expression using shRNA results in B) decreased invasive behavior (^p < 0.0001 relative to Scrambled shRNA control (Scram)), C) reduced ability to induce endothelial cell network formation (^p < 0.0001 relative to control (Scram)) and, D) significantly slower (^p = 0.01) tumor xenograft growth in female, athymic nude mice relative to scrambled shRNA transfected cells (MDA-MB-231-SHH-Scram). E) Immunohistochemical analysis of the primary tumor xenograft tissue for CYR61 (Panels i and ii), angiogenic microvessels (Panels iii and iv), SHH (Panels v and vi) (p > 0.05) and Ki67 (Panels vii and viii). Shown are representative images for control (Scram) and MDA-MB-231-SHH cells stably transfected with shRNA to CYR61. F) The xenografts obtained from MDA-MB-231-SHH-shCYR61 cells show lower immunoreactive scores for CYR61, microvessel density, MVD (^p = 0.0006) and Ki67 (^p = 0.0062). The levels of SHH expression were comparable between the control (Scram) and cells silenced stably for CYR61. G) The incidence of spontaneous pulmonary metastasis is decreased in CYR61-silenced SHH-expressing MDA-MB-231 cells (^p =0.01).

Article Snippet: SHH and CYR61 mRNA expression in human breast cancer patient tissues was analyzed using tissue qPCR array cDNA samples from OriGene Technologies (Rockville, MD). qPCR was performed per the manufacturers’ recommendations.

Techniques: Expressing, shRNA, Transfection, Immunohistochemical staining, Stable Transfection

Journal: Oncogene

Article Title: INCREASED VASCULARITY AND SPONTANEOUS METASTASIS OF BREAST CANCER BY HEDGEHOG SIGNALING MEDIATED UPREGULATION OF CYR61

doi: 10.1038/onc.2011.496

Figure Lengend Snippet: We assessed the transcript levels of SHH and CYR61 in a panel of tissues encompassing normal breast tissue (n=4) and breast tumor tissues by QRT-PCR. The panel included tissues representing Stage 1 (n=2), Stage 2 (n=24) and Stage 3 & 4 (n=21). A) Overall, the transcript levels of SHH are elevated in breast tumors derived from advanced stage breast cancer (^p = 0.0027). B) The transcript levels of CYR61 are significantly higher in advanced stage breast cancer relative to normal tissues (^p = 0.0011). The data is represented as relative transcript levels of SHH and CYR61 normalized to β-actin.

Article Snippet: SHH and CYR61 mRNA expression in human breast cancer patient tissues was analyzed using tissue qPCR array cDNA samples from OriGene Technologies (Rockville, MD). qPCR was performed per the manufacturers’ recommendations.

Techniques: Quantitative RT-PCR, Derivative Assay

Journal: Oncogene

Article Title: miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer

doi: 10.1038/onc.2013.381

Figure Lengend Snippet: A, Patients with DAB2 mRNA levels in the highest and lowest quintiles were selected from the 245 NSCLC patients, defining the high and low DAB2 expression groups (n=50/group). The mean DAB2 mRNA levels are significantly different between the two patient groups. ****, p < 0.0001. B , Kaplan-Meier overall survival curves for the two groups of NSCLC patients. Median overall survival times in the low (green) and high DAB2 (red) groups were 4.2 y and > 10.2 y, respectively, with a hazard ratio of 0.36 (95% CI 0.16–0.69), and p < 0.01. C , Recurrence-free survival curves for the two groups. Median recurrence-free survival times in the low and high DAB2 groups were 2.5 y and > 7.5 y, respectively, with a hazard ratio of 0.53 (95% CI 0.29–0.96), and p = 0.04. Statistical significance was assessed by Mantel-Cox log-rank test.

Article Snippet: The coding region of the DAB2 mRNA with and without the 3′UTR was amplified by PCR from human genomic DNA and a DAB2 human cDNA clone (Origene, Rockville, MD), respectively.

Techniques: Expressing

Journal: Oncogene

Article Title: miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer

doi: 10.1038/onc.2013.381

Figure Lengend Snippet: Expression levels of DAB2 mRNA levels were measured using Illumina Human WG-6 V3 expression arrays in three groups of cell lines – SCLC (n=29), NSCLC (n=96), and immortalized normal lung cells (n=59) which include normal human bronchial epithelial cells (HBECs) and human small airway epithelial cells (HSAECs). After background subtraction and normalization of the raw intensity data, the DAB2 mRNA expression levels were calculated as relative signal intensity. Statistical significance was assessed by student t-test. *, p < 0.05; ****, p < 0.0001.

Article Snippet: The coding region of the DAB2 mRNA with and without the 3′UTR was amplified by PCR from human genomic DNA and a DAB2 human cDNA clone (Origene, Rockville, MD), respectively.

Techniques: Expressing

Journal: Oncogene

Article Title: miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer

doi: 10.1038/onc.2013.381

Figure Lengend Snippet: A, H1993 cells were stably transfected with either pcDNA3.1 vector containing DAB2 or a null pcDNA3.1 vector without a DAB2 insert. Expression levels of Dab2 protein were detected by western blot using an anti-Dab2 antibody, with β-actin levels used as a loading control. B, BrdU incorporation assay as a function of DAB2 over-expression. C, Retroviral pGFP-V-RA-DAB2-shRNA expression constructs were used to knock down endogenous DAB2 expression. H1993 cell lines with stably integrated DAB2-shRNA expression or control shRNA constructs were generated as described in the Materials and Methods. Expression levels of Dab2 protein were detected as above. D, BrdU incorporation as a function of DAB2 knockdown. E, Cell cycle analysis as a function of DAB2 knockdown. The fractions of cells in G 1 , S and G 2 phases were estimated using the Watson pragmatic model. Similar results were obtained from three independent experiments. *, p < 0.05; **, p < 0.01.

Article Snippet: The coding region of the DAB2 mRNA with and without the 3′UTR was amplified by PCR from human genomic DNA and a DAB2 human cDNA clone (Origene, Rockville, MD), respectively.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Western Blot, BrdU Incorporation Assay, Over Expression, shRNA, Construct, Generated, Cell Cycle Assay

Journal: Oncogene

Article Title: miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer

doi: 10.1038/onc.2013.381

Figure Lengend Snippet: Expression of miR-93 — but not other miRNAs that target DAB2 — is significantly anti-correlated with DAB2 expression in lung tumor specimens Using a TargetScan P CT score > 0.1 as a cutoff, 9 miRNAs were identified to target the 3′UTR of DAB2. (1), TargetScan predictions, showing the predicted context score, probability of conserved targeting (P CT ) score, and the prediction rank based on the P CT score ( 23 , 24 , 42 ). (2), miRmate predictions showing the predicted interaction score, as described previously ( 19 ). (3), The correlations between miRNAs and DAB2 mRNA levels were assessed by Pearson correlation, with p < 0.05 considered statistically significant.

Article Snippet: The coding region of the DAB2 mRNA with and without the 3′UTR was amplified by PCR from human genomic DNA and a DAB2 human cDNA clone (Origene, Rockville, MD), respectively.

Techniques: Expressing

Journal: Oncogene

Article Title: miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer

doi: 10.1038/onc.2013.381

Figure Lengend Snippet: A, The predicted interaction of miR-93 and the candidate target site in the DAB2 3′UTR with the predicted free energy of hybridization. The seed sequence is highlighted in red. B, H1993 cells were co-transfected with the indicated vectors. After 48 h of transfection, cells were lysed and luciferase activity was measured. Shown are normalized luciferase activities of different treatment groups. Differences in luciferase activity were assessed by two-tailed t-test. ***, p < 0.001.

Article Snippet: The coding region of the DAB2 mRNA with and without the 3′UTR was amplified by PCR from human genomic DNA and a DAB2 human cDNA clone (Origene, Rockville, MD), respectively.

Techniques: Hybridization, Sequencing, Transfection, Luciferase, Activity Assay, Two Tailed Test

Journal: Oncogene

Article Title: miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer

doi: 10.1038/onc.2013.381

Figure Lengend Snippet: A, H1993 cells were transiently transfected with equal amounts of either control pcDNA3.1 vectors, pcDNA3.1-DAB2-3′UTR, pCMV6-miR-93, pcDNA3.1-DAB2-3′UTR/pCMV6-miR-93, or pcDNA3.1-DAB2/pCMV6-miR-93 as indicated. Cell lysates were harvested after 48 hrs. Protein expression levels of Dab2, total Akt, and pAkt were measured by western blot, with β-actin as a loading control. B, H1993 cells were transfected as above. After 48 hrs, cell proliferation was measured by BrdU incorporation assay. C–D, Effect of miR-93 and DAB2 over-expression on Dab2 and Akt protein expression ( C ) and on cell proliferation ( D ) in SCLC cell line H524, as above. E, Effect of Akt kinase inhibitor and DAB2 over-expression on Dab2 and Akt protein expression. F, Effect of Akt kinase inhibitor and DAB2 over-expression on cell proliferation in H1993 cells. G–H , Effect of DAB2 over-expression ( F ) or knockdown ( G ) on cell viability in a panel of lung cancer cell lines. I–J, Effect of miR-93 over-expression ( I ) or knockdown ( J ) on cell viability in a panel lung cancer cell lines. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The coding region of the DAB2 mRNA with and without the 3′UTR was amplified by PCR from human genomic DNA and a DAB2 human cDNA clone (Origene, Rockville, MD), respectively.

Techniques: Transfection, Expressing, Western Blot, BrdU Incorporation Assay, Over Expression

Journal: Oncogene

Article Title: miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer

doi: 10.1038/onc.2013.381

Figure Lengend Snippet: mRNA levels of DAB2 and FUS1 — but not other miR-93 targets — are significantly inversely correlated with miR-93 expression levels in lung tumor specimens Correlations between miR-93 levels and those of previously identified miR-93 target genes were assessed in lung tumor specimens by Pearson correlation. Shown are the correlation coefficients (r) and p values.

Article Snippet: The coding region of the DAB2 mRNA with and without the 3′UTR was amplified by PCR from human genomic DNA and a DAB2 human cDNA clone (Origene, Rockville, MD), respectively.

Techniques: Expressing

Journal: Oncogene

Article Title: miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer

doi: 10.1038/onc.2013.381

Figure Lengend Snippet: Heatmap presentation of the expression of miR-93 and DAB2 mRNA in 45 cell lines, with blue indicating relative under-expression and yellow indicating relative over-expression. The correlation between miR-93 and DAB2 mRNA levels was assessed by Pearson correlation.

Article Snippet: The coding region of the DAB2 mRNA with and without the 3′UTR was amplified by PCR from human genomic DNA and a DAB2 human cDNA clone (Origene, Rockville, MD), respectively.

Techniques: Expressing, Over Expression

Journal: Oncogene

Article Title: miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer

doi: 10.1038/onc.2013.381

Figure Lengend Snippet: A, Mice injected with H1993 cells stably over-expressing miR-93 or control H1993 cells with stably integrated empty vector. Images were taken at the end point of the experiment. B, Tumors in each group. C, Time-dependent tumor growth curves of the different treatment groups. D, Tumor levels of miR-93, measured by qRT-PCR. E, Tumor levels of Dab2 and Ki67 proteins, measured by western blot. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The coding region of the DAB2 mRNA with and without the 3′UTR was amplified by PCR from human genomic DNA and a DAB2 human cDNA clone (Origene, Rockville, MD), respectively.

Techniques: Injection, Stable Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: Oncogene

Article Title: miR-93-directed down-regulation of DAB2 defines a novel oncogenic pathway in lung cancer

doi: 10.1038/onc.2013.381

Figure Lengend Snippet: miR-93 targets multiple genes and DAB2 is a target of multiple miRNAs. Among the miRNAs that target DAB2, miR-93 plays the major role in down-regulating DAB2 expression in lung cancer cells. Reciprocally, DAB2 is the primary target that mediates the growth-promoting function of miR-93 in lung cancer cells. †, predicted interactions that have not been experimentally validated.

Article Snippet: The coding region of the DAB2 mRNA with and without the 3′UTR was amplified by PCR from human genomic DNA and a DAB2 human cDNA clone (Origene, Rockville, MD), respectively.

Techniques: Expressing