NIH-3T3 murine fibroblast transfection and MEF mouse embryonic fibroblast transfection using DreamFect.

NIH-3T3 and MEF fibroblasts were transfected using DreamFect lipid-based transfection reagent.

Paper reference: Cell Death Differ. 2012 Oct 12. doi: 10.1038/cdd.2012.129.

Phosphorylation of Atg5 by the Gadd45β-MEKK4-p38 pathway inhibits autophagy.

Keil E, Höcker R, Schuster M, Essmann F, Ueffing N, Hoffman B, Liebermann DA, Pfeffer K, Schulze-Osthoff K, Schmitz I.

Abstract

Autophagy is a lysosomal degradation pathway important for cellular homeostasis, mammalian development, cancer and immunity. Many molecular components of autophagy have been identified, but little is known about regulatory mechanisms controlling their effector functions. Here, we show that, in contrast to other p38 MAP kinase activators, the growth arrest and DNA damage 45 beta (Gadd45β)-MAPK/ERK kinase kinase 4 (MEKK4) pathway specifically directs p38 to autophagosomes. This process results in an accumulation of autophagosomes through p38-mediated inhibition of lysosome fusion. Conversely, autophagic flux is increased in p38-deficient fibroblasts and Gadd45β-deficient cells. We further identified the underlying mechanism and demonstrate that phosphorylation of the autophagy regulator autophagy-related (Atg)5 at threonine 75 through p38 is responsible for inhibition of starvation-induced autophagy. Thus, we show for the first time that Atg5 activity is controlled by phosphorylation and, moreover, that the spatial regulation of p38 by Gadd45β/MEKK4 negatively regulates the autophagic process.Cell Death and Differentiation advance online publication, 12 October 2012; doi:10.1038/cdd.2012.129.

DreamFect Gold from OZ Biosciences allows transfecting all types of nucleic acids with a very high efficiency; it is fully biodegradable and does not interfere with cellular mechanisms.