Journal: PLoS ONE

Article Title: p120RasGAP Is a Mediator of Rho Pathway Activation and Tumorigenicity in the DLD1 Colorectal Cancer Cell Line

doi: 10.1371/journal.pone.0086103

Figure Lengend Snippet: A) Chromatogram showing relative intensities of each base pair after Sanger sequencing in both the genomic and cDNA derived from the cell lines. B) Illustration of location of the mutation at the RasGAP protein level. C) RASA1 expression data derived from all cell lines in the Broad Institute Cancer Cell Line Encyclopedia. Bars represent mean.

Article Snippet: Entry vector containing either CMV promoter or RASA1 ORF (OpenBiosystems, ThermoScientific, Ottawa, ON) were recombined with pLenti-CMV-GFP-DEST vector (Addgene plasmid 19732) creating pLentiCMV and pLentiRASA1.

Techniques: Sequencing, Derivative Assay, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: p120RasGAP Is a Mediator of Rho Pathway Activation and Tumorigenicity in the DLD1 Colorectal Cancer Cell Line

doi: 10.1371/journal.pone.0086103

Figure Lengend Snippet: Wild-type (WT) or mutant KRAS was overexpressed in DKO4 cells. mRNA was extracted from cells and quantified using rt-qPCR to measure KRAS (A) or RASA1 (B). C) Western blotting showing levels of these proteins, along with activation status of KRAS. Correlation of mRNA (D) and protein expression using densitometry analysis of Western blotting (E) of KRAS and RASA1 after knockdown of KRAS using 11 different shRNAs. For protein correlation, outliers over 3 standard deviations from the mean were excluded. All quantification is relative to empty vector. Statistical analysis of expression using unpaired t-test, ***p<0.001, *p<0.05.

Article Snippet: Entry vector containing either CMV promoter or RASA1 ORF (OpenBiosystems, ThermoScientific, Ottawa, ON) were recombined with pLenti-CMV-GFP-DEST vector (Addgene plasmid 19732) creating pLentiCMV and pLentiRASA1.

Techniques: Mutagenesis, Quantitative RT-PCR, Western Blot, Activation Assay, Expressing, Plasmid Preparation

Journal: PLoS ONE

Article Title: p120RasGAP Is a Mediator of Rho Pathway Activation and Tumorigenicity in the DLD1 Colorectal Cancer Cell Line

doi: 10.1371/journal.pone.0086103

Figure Lengend Snippet: A) Cell counting assay in RasGAP overexpressing and knockdown cells. B) Cell adhesion to collagen. Statistical significance was determined by t-test: *p<0.05, ***p<0.001 C) Wound healing assay. D) Rhodamine-phalloidin staining of actin filaments after overnight adhesion to collagen. E) Tumor volume and (F) final excised tumor weight of xenograft tumors in SCID mice derived from subcutaneous injection of DLD1 empty vector (GFP), DKO4 GFP, or DKO4 overexpressing RasGAP. Number of mice used is indicated on graph. p-value calculated as indicated in section. G) rt-qPCR analysis of RASA1 gene expression derived from xenograft tumors after excision.

Article Snippet: Entry vector containing either CMV promoter or RASA1 ORF (OpenBiosystems, ThermoScientific, Ottawa, ON) were recombined with pLenti-CMV-GFP-DEST vector (Addgene plasmid 19732) creating pLentiCMV and pLentiRASA1.

Techniques: Cell Counting, Wound Healing Assay, Staining, Derivative Assay, Injection, Plasmid Preparation, Quantitative RT-PCR, Expressing

Journal: Cell Transplantation

Article Title: Improvement of Heart Function After Transplantation of Encapsulated Stem Cells Induced with miR-1/Myocd in Myocardial Infarction Model of Rat

doi: 10.1177/09636897211048786

Figure Lengend Snippet: Transduction efficiency of MSCs with lentiviruses containing miR-1 and Myocd. (A) Transduced cells were observed by fluorescence microscopy regarding expression of GFP reporter. Moreover, percentage of transduced cells was measured by flow cytometry. About 80% of MSCs were transduced compared to the control cells. ( B) Expression of miR-1 and Myocd in different groups was evaluated by qRT-PCR. ** P < 0.01, *** P < 0.001; data were expressed as mean ± SD, n = 3. (C) Cell viability of transduced cells was studied by MTT analysis. In intervention groups, cell viability was significantly reduced on days 14 and 21. * P < 0.05, ** P < 0.01, *** P < 0.001. GFP: Green fluorescent protein.

Article Snippet: The mmu-mir-1 vector (containing the cytomegalovirus (CMV) and Simian virus 40(SV40) promoters) (Applied Biological Materials, Canada, mm10023), as well as Myocd (abm, 3130606) and pLenti-III-miR-GFP-Blank (abm, m001) as control were separately co-transfected with psPAX2 (containing gag/pol element) and pMD2.

Techniques: Transduction, Fluorescence, Microscopy, Expressing, Flow Cytometry, Quantitative RT-PCR

Journal: Cell Transplantation

Article Title: Improvement of Heart Function After Transplantation of Encapsulated Stem Cells Induced with miR-1/Myocd in Myocardial Infarction Model of Rat

doi: 10.1177/09636897211048786

Figure Lengend Snippet: Differentiation of transduced cells into iCMs. ( A) Seven days after transduction, morphology of MSC miR-1 and MSC miR-1/Myocd groups became spindle-shaped or star-shaped. Twenty-one days after transduction, the cells became more differentiated and matured and short spindle-shaped or polygonal cells were observed, similar to cardiomyocytes. ( B) qRT-PCR analysis was performed for cardiac specific markers of Tnnt2 , Nkx2-5 , and Gata4 . Expression of markers was compared in MSC miR-1 and MSC miR-1/Myocd groups with MSC null as a negative control. The results showed that expression of Tnnt2 , Nkx2-5 , and Gata4 was increased significantly in MSC miR-1/Myocd group in comparison with other groups, on days 7, 14, and 21 after transduction. * P < 0.05, ** P < 0.01, *** P < 0.001; Transcript value was shown as mean±SD. ( C) ICC analysis detected cTnT, Nkx2-5, and GATA4 markers in MSC miR-1 and MSC miR-1/Myocd groups on day 21. Nucleus staining was conducted by DAPI stain. An Olympus fluorescence microscope was used to record images.

Article Snippet: The mmu-mir-1 vector (containing the cytomegalovirus (CMV) and Simian virus 40(SV40) promoters) (Applied Biological Materials, Canada, mm10023), as well as Myocd (abm, 3130606) and pLenti-III-miR-GFP-Blank (abm, m001) as control were separately co-transfected with psPAX2 (containing gag/pol element) and pMD2.

Techniques: Transduction, Quantitative RT-PCR, Expressing, Negative Control, Staining, Fluorescence, Microscopy

Journal: Cell Transplantation

Article Title: Improvement of Heart Function After Transplantation of Encapsulated Stem Cells Induced with miR-1/Myocd in Myocardial Infarction Model of Rat

doi: 10.1177/09636897211048786

Figure Lengend Snippet: Differentiation of transduced cells into iCM after encapsulation in the CS/CO hydrogel. Three days after co-transduction of MSCs with miR-1 and Myocd lentiviruses, the infected cells were trypsinized and embedded in the CS/CO hydrogel. ( A) Expression of Tnnt2 , Nkx2-5 , and Gata4 , and was evaluated by qRT-PCR in 2D and 3D cell cultures (CS/CO hydrogel). Expression of markers was increased significantly in the 3D cell culture in comparison with the 2D cell culture. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; Transcript value was shown as mean±SD. ( B) Immunofluorescence staining of transduced cells 21 days after transduction showed that expression of the markers was enhanced in 3D culture. iCMs: Induced cardiomyocytes, CS/CO: Chitosan/collagen, 3D culture: Three dimensional culture.

Article Snippet: The mmu-mir-1 vector (containing the cytomegalovirus (CMV) and Simian virus 40(SV40) promoters) (Applied Biological Materials, Canada, mm10023), as well as Myocd (abm, 3130606) and pLenti-III-miR-GFP-Blank (abm, m001) as control were separately co-transfected with psPAX2 (containing gag/pol element) and pMD2.

Techniques: Transduction, Infection, Expressing, Quantitative RT-PCR, Cell Culture, Immunofluorescence, Staining