HEK-293, MCF7, HeLa and MDA-MB-231 were transfected with 1 µg DNA per 1 million cells using DreamFect at the recommended conditions.
This paper shows the efficiency of DreamFect transfection reagent from OZ Biosciences to transfect cell lines with plasmid DNA.article reference: Biometals. 2014 May 14.
Delta-lactoferrin induces cell death via the mitochondrial death signaling pathway by upregulating bax expression.
Abstract
Delta-lactoferrin (∆Lf) is a transcription factor belonging to the lactoferrin family, the expression of which inhibits cell proliferation and leads to Skp1 and DcpS gene transactivation. In this study, we showed that ∆Lf expression also induces cell death via apoptosis in HEK 293 and MCF7 cells using a cell
viability assay and DNA fragmentation. Western blot analyses showed
that apoptosis was caspase-9, 7 and 8 dependent. Proteolytic cleavage of
the endonuclease PARP was significantly increased. The levels of
expression of Bcl family members were detected by immunochemistry and
showed that the Bcl-xl/Bax and Bcl-2/Bax protein ratios were decreased.
We determined that the pro-apoptotic effects of ∆Lf are mainly mediated
by the activation of the mitochondria-dependent death-signaling pathway.
Apoptosis induction by ∆Lf is concomitant with increased cellular
levels of Bax protein. Analysis of the Bax promoter region detected a
∆Lf response element located at -155 bp from the transcription start
site. Both luciferase reporter gene and chromatin immunoprecipitation
assays confirmed that ∆Lf interacts in vitro and in vivo specifically
with this sequence. Its deletion, realized using directed mutagenesis,
totally abolished ∆Lf transcriptional activity, identifying it as a
∆Lf-responsive element. These results indicate that the Bax gene is a
novel ∆Lf target. Moreover we also showed that the O-GlcNAc/P interplay,
which controls ∆Lf transcriptional activity, modulates Bax
transactivation.
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