empty control vector pcineo  (Promega)

Journal: Scientific Reports

Article Title: Krüppel-like factor 6 is a transcriptional activator of autophagy in acute liver injury

doi: 10.1038/s41598-017-08680-w

Figure Lengend Snippet: KLF6 transcriptionally activates autophagy-related genes in a p53-dependent manner. Transfection with the KLF6 -expression vector pcIneo-KLF6 induced KLF6 mRNA ( A ) and protein ( B ) expression. KLF6-over-expression induces autophagy in HepG2 cells as assessed by LC3 Western blotting ( C ) and quantification of LC3-II-bands normalized to loading control GAPDH ( D ; fold change versus control shown as mean ± SEM of n = 3 independent cell culture experiments; full length Western blot images are given in Supplementary information.). Autophagic flux was assessed by LC3-II and p62 Western blotting in HepG2 and pcIneo-KLF6 transfected cells in absence (−) or presence ( + ) of 100 µM chloroquine for 24 h (( C ) shows representative Western blot images, ( D) shows fold change versus control, shown as mean ± SEM of n = 3 independent experiments). Activation of the ATG7 and BECLIN1 promoter was quantified by luciferase activity in a co-transfection experiment of pcIneo-KLF6 with specific promoter reporter luciferase plasmids in HepG2 ( E ) and in p53-deficient HepG2-303 cells ( F ). The interaction of KLF6 with the ATG7 and BECLIN1 promoter containing putative KLF6-binding sites was confirmed by chromosomal immunoprecipitation (ChIP) in HepG2 cells ( G ) using two different KLF6 antibodies, IgG as negative controls, Histone-H3 antibody was used as a positive control for ChIP; full length images of agarose gels are given in Supplementary information.

Article Snippet: KLF6-expression plasmid pcIneo-KLF6 or empty control vector pcIneo (Promega, Madison, WI, USA) were used at 80 ng per 3.9 cm 2 well.

Techniques: Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot, Cell Culture, Activation Assay, Luciferase, Activity Assay, Cotransfection, Binding Assay, Immunoprecipitation, Positive Control

Journal: Scientific Reports

Article Title: Krüppel-like factor 6 is a transcriptional activator of autophagy in acute liver injury

doi: 10.1038/s41598-017-08680-w

Figure Lengend Snippet: Autophagosome formation in KLF6 over-expressing HepG2 cells. Under a transmission electron microscope, the autophagosome formation was observed and imaged in HepG2 cells transfected with the empty vector (pcIneo) ( A ), in HepG2 cells transfected with pcIneo treated with 15 µM rapamycin for 6 h to stimulate autophagosome formation ( B ) and in KLF6 -over-expressing HepG2 cells (pcIneo-KLF6) ( C ). Representative slides and blow-ups shown are shown of n = 2 independent cell culture experiments.

Article Snippet: KLF6-expression plasmid pcIneo-KLF6 or empty control vector pcIneo (Promega, Madison, WI, USA) were used at 80 ng per 3.9 cm 2 well.

Techniques: Expressing, Transmission Assay, Microscopy, Transfection, Plasmid Preparation, Cell Culture

Journal: Scientific Reports

Article Title: Krüppel-like factor 6 is a transcriptional activator of autophagy in acute liver injury

doi: 10.1038/s41598-017-08680-w

Figure Lengend Snippet: Autophagosome formation in KLF6 over-expressing HepG2 cells. To visualize formation of autophagosomes in HepG2 cells transfected with empty control vector (pcIneo) or in KLF6 over-expressing HepG2 cells (pcIneoKLF6) we treated the cells with Autophagy Tandem Sensor RFP-GFP-LC3B. As a positive control, we stimulated autophagosome formation via treatment with 15 µM rapamycin for 6 h. Cells were viewed and imaged with a Leica SP8 confocal microscope (20-fold magnification); shown are representative images of n = 3 individual cell culture experiments.

Article Snippet: KLF6-expression plasmid pcIneo-KLF6 or empty control vector pcIneo (Promega, Madison, WI, USA) were used at 80 ng per 3.9 cm 2 well.

Techniques: Expressing, Transfection, Plasmid Preparation, Positive Control, Microscopy, Cell Culture