Journal: eLife

Article Title: Repurposing of KLF5 activates a cell cycle signature during the progression from a precursor state to oesophageal adenocarcinoma

doi: 10.7554/eLife.57189

Figure Lengend Snippet: ( A ) Western blots of the indicated proteins or phosphorylated ( p ) sites in CP-A cells stably transfected with empty or ERBB2 expressing vectors grown in serum free medium for 48 hr (lanes 1 and 2) or serum-free media and stimulated with complete media for 15 min (lanes 3 and 4). ( B ) Relative growth of CP-A cells stably transfected with empty or ERBB2 expressing vectors grown in serum-free media for 6 days (n = 3; ** = p value<0.01, two-way ANOVA). ( C ) RT-qPCR analysis of the indicated genes (normalised to GAPDH ) in serum starved CP-A cells stably transfected with empty or ERBB2 expressing vectors (n = 3; p-values, *=<0.05, **=<0.01). ( D ) ChIP-qPCR of KLF5 binding to regulatory regions associated with the indicated genes in CP-A cells stably transfected with empty or ERBB2 expressing vectors grown in serum-free media for 48 hr. Non-specific IgG is provided as a control (n = 3; ns = non-significant increases).

Article Snippet: Transfected construct (human) , Full length guide RNAs targeting KLF5 TSS , Synthego , , 5’-GUGCGCUCGCGGUUCUCUCG-3’ 5’-AGGACGUUGGCGUUUACGUG-3’ 5’-GCGUCAAGUGUCAGUAGUCG-3’.

Techniques: Western Blot, Stable Transfection, Transfection, Expressing, Quantitative RT-PCR, ChIP-qPCR, Binding Assay, Control

Journal: eLife

Article Title: Repurposing of KLF5 activates a cell cycle signature during the progression from a precursor state to oesophageal adenocarcinoma

doi: 10.7554/eLife.57189

Figure Lengend Snippet:

Article Snippet: Transfected construct (human) , Full length guide RNAs targeting KLF5 TSS , Synthego , , 5’-GUGCGCUCGCGGUUCUCUCG-3’ 5’-AGGACGUUGGCGUUUACGUG-3’ 5’-GCGUCAAGUGUCAGUAGUCG-3’.

Techniques: Transfection, Plasmid Preparation, Control, Stable Transfection, Isolation, Construct, Western Blot, Recombinant, Sequencing, SYBR Green Assay, Concentration Assay, Software

Journal: Biotechnology and bioengineering

Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

doi: 10.1002/bit.27561

Figure Lengend Snippet: Knockout of the CASP8AP2 gene. (a) Agarose gel electrophoresis of polymerase chain reaction (PCR) amplified CASP8AP2 locus in the parental and the 892–7 cell lines, and EMBOSS WATER sequence alignment of both alleles with the parental sequence. Guide RNA (gRNA) target sequence is highlighted. GAPDH-normalized qPCR analysis of CASP8AP2 messenger RNA (mRNA) abundance in parental and CASP8AP2 knockout clone, 892–7, using primers targeting the gRNA locus for amplification. (b) Caspase 8 activity in cell lysates of control (uninduced) and apoptosis-induced (induced) 892–7 and parental cells (**p < .001)

Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

Techniques: Knock-Out, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Sequencing, Activity Assay, Control

Journal: Biotechnology and bioengineering

Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

doi: 10.1002/bit.27561

Figure Lengend Snippet: Luciferase and secreted alkaline phosphatase (SEAP) production from the parental cell line and the CASP8AP2 knockout clone, 892–7. (a) Luciferase enzymatic activity (***p < .0001). (b) GAPDH-normalized luciferase messenger RNA (mRNA) abundance determined using real-time quantitative polymerase chain reaction (** p < .001). (c) Luciferase expression by Western blot analysis. (d) Alkaline phosphatase activity in the cell culture supernatant of cells transfected with a plasmid encoding the SEAP gene (*p < .05). Results represent the average of at least three biological replicates, and error bars represent the standard deviation

Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

Techniques: Luciferase, Knock-Out, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Cell Culture, Transfection, Plasmid Preparation, Standard Deviation

Journal: Biotechnology and bioengineering

Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

doi: 10.1002/bit.27561

Figure Lengend Snippet: The effect of CASP8AP2 knockout on cell cycle progression by FACS-based, propidium iodide cell cycle analysis. (a) Knockout clone. (b) Parental cells. (c) Percentages of cells in the G1, S, or G2/M in asynchronous cultures calculated using BD FACS DIVA software. Results represent the average of three analyses, and error bars represent the standard deviation (*p < .05). (d) Parental and 892–7 cells were treated with thymidine to synchronize the cell cycle. After release from the thymidine block, the cells were harvested at the indicated time points and cell cycle progression was analyzed using a cytometer

Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

Techniques: Knock-Out, Cell Cycle Assay, Software, Standard Deviation, Blocking Assay, Cytometry

Journal: Biotechnology and bioengineering

Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

doi: 10.1002/bit.27561

Figure Lengend Snippet: Summary of raw and processed RNA-seq reads generated from CASP8AP2 mutant clone, 892–7, and parental RNA samples

Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

Techniques: Generated, Mutagenesis

Journal: Biotechnology and bioengineering

Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

doi: 10.1002/bit.27561

Figure Lengend Snippet: Pathway ranking based on Z-score following enrichment analysis of the differentially expressed genes between the CASP8AP2 knockout and the parental cell lines. Gray bars represent pathways involved in the cell cycle

Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

Techniques: Knock-Out

Journal: Biotechnology and bioengineering

Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

doi: 10.1002/bit.27561

Figure Lengend Snippet: (a and b) Relative CDKN2A and HDAC10 expression in parental and CASP8AP2 knockout HEK293 cells determined in a GAPDH-normalized real-time quantitative polymerase chain reaction. (c) Effect of overexpressing CDKN2A on luciferase activity in parental (parental CDKN2A) and CASP8AP2 knockout (892–7 CDKN2A) HEK293 cells (*p < .05). (d) Effect of CDKN2A overexpression on cell growth of the parental cell line. Results represent the average of at least three biological replicates, with error bars representing standard deviation

Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

Techniques: Expressing, Knock-Out, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Over Expression, Standard Deviation

Journal: Biotechnology and bioengineering

Article Title: Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene

doi: 10.1002/bit.27561

Figure Lengend Snippet: Identification of the CDKN2A gene as an effector for recombinant protein expression in the CASP8AP2 deficient 892–7 clone

Article Snippet: CRISPR/Cas9 constructs Two plasmids encoding paired guide RNAs (gRNAs) targeting the 10th exon of the CASP8AP2 gene, designed by transOMIC Technologies, were used in this study.

Techniques: Recombinant, Expressing