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: onali@unica.it
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.