Article:
“Interferon-β counter-regulates its own pro-apoptotic action by activating
p38 MAPK signalling in human SH-SY5Y neuroblastoma cells
Journal :
Apoptosis
Authors:
Simona Dedoni, Maria C. Olianas and Pierluigi Onali
Corresponding author: Pierluigi Onali, Laboratory of Cellular and Molecular Pharmacology, Section of Neurosciences and Clinical Pharmacology, Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato (Ca), Italy.
E-mail address:
Supplementary material
Online Resource 1. The pan-JAK inhibitor P6 inhibits IFN-β-induced ROS generation. SH-SY5Y cells were pretreated with either vehicle or P6 (2 µM) for 6h and then exposed to either vehicle or IFN-β (30 ng/ml) for 6 h. Thereafter, cells were loaded with DCFDA and the emitted fluorescence was determined by using a spectrophotometer plate reader as described under Materials and methods. Values are expressed as percent of control and are the mean ± S.E.M. of three separate experiments. * p < 0.05 vs control, # p < 0.05 by ANOVA followed by Newman-Keuls post hoc test.
Online Resource. 2. Effects of IFN-β on SH-SY5Y cell viability. Cells were grown to confluency and then treated for the indicated time periods with IFN-β (30 ng/ml). Cells were then detached, washed and incubated with Cell count and Viability reagent (Millipore) according to the manufacturer’s instruction. The percent of viable cells was then determined by using Muse Cell Analyzer (Count and Viability Software) (Millipore). Values are the mean ± S.E.M. of four separate experiments run in triplicate. *** p < 0.001.
Online Resource 3. Effects of the ROS scavenger N-acetyl-L-cysteine (NAC) and the TAK1 inhibitor 5Z-oxozeaenol (oxoz) on IFN-β-induced PARP cleavage. a, SH-SY5Y cells were treated for 24 h with either vehicle, IFN-β (30 ng/ml), NAC (1 mM) or NAC + IFN-β. Cell lysates were analyzed for cleaved PARP, PARP and actin. Values are the mean ± S.E.M. of four experiments. b, cells were pre-treated with either vehicle or oxoz (100 nM) for 30 min and then exposed to either vehicle or IFN-β (30 ng/ml) for 24 h. Cell lysates were analyzed for cleaved PARP, PARP and actin. Values are the mean ± S.E.M. of three experiments. *** p < 0.001 vs control (vehicle alone), # p < 0.05 by ANOVA followed by Newman-Keuls post hoc test.
Online Resource 4. Effects of IFN-β on JNK phosphorylation. a, SH-SY5Y cells were exposed to either vehicle or IFN-β (30 ng/ml) for the indicated periods of time. Control samples (0 time) were treated with vehicle. Cell lysates were then analyzed for the levels of phospho-JNK (p-JNK), total JNK and actin by Western blot. Values are the mean ± S.E.M. of four experiments. * p < 0.05 vs control. b, cells were treated with either control siRNA or p38α MAPK (p38α) siRNA and then exposed for 24 h to either vehicle or IFN-β (30 ng/ml). Cell lysates were analyzed for phospho-JNK, total JNK, p38 MAPK and actin. Values of phospho-JNK levels are expressed as percent of control (control siRNA + vehicle) and are the mean ± S.E.M. of three separate experiments. *** p < 0.001, * p < 0.05 vs control, # p < 0.05 by ANOVA followed by Newman-Keuls post hoc test.