The changes in the cell antioxidant (SOD, GPx) and oxidant (H2O2) enzyme activities were measured using respective cellular activity assay kits (Jiancheng Biochemical Co., Ltd., Nanjing, China).
We previously shed light on the importance of the N-terminus of TDP-43: we have shown that deletion of the first 75 amino acid residues of TDP-43 significantly reduces its biological activity, as measured using a cellular cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 splicing assay (14).
The cell glutathione peroxidase (GPx) activity was measured using the GPx cellular activity assay kit from Jiancheng Biochemical Co., Ltd.
After treatment, Calcineurin activity was measured using a Calcineurin cellular activity assay kit (Enzo Life Sciences Inc., Farmingdale, NY, USA), following manufacturer's instructions.
Cell proliferation was measured using xCELLigence Real Time Cellular Analysis (RTCA) system (Roche, Germany), as previously described [ 19].
Staining intensity was measured using a Chromavision Automated Cellular Imaging System (Chromavision® ACIS®, San Juan Capistrano, CA) as previously described [ 12].
An Ara-C dependent increase was seen in the fraction of apopotic cells on flowcytometry and a concentration dependent decline was seen in the cellular viability measured using XTT reagent.
A total of 40 μg cellular protein (measured using the Bradford assay and bovine serum albumin as standards) was loaded into sodium dodecyl sulphate polyacrylamide gel electrophoresis under nonreducing conditions.
Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer.
Cellular viability was examined by counting the viable cells using trypan blue dye exclusion, and cellular proliferation was measured using an MTS proliferation assay kit (Promega, Madison, WI, USA).
