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Slattery et al. Neuroimmunol Neuroinflammation 2018;5:11 I http://dx.doi.org/10.20517/2347-8659.2018.05 Page 11 of 15

150

Nitrite (% control) 100

50

0
0 2 20 50
P-aminophenol (µmol/L)

0 µmol/L BCTC 0.5 µmol/L BCTC
0.02 µmol/L BCTC 2 µmol/L BCTC

Figure 10. Effects of p-aminophenol alone or in combination with the TRPV1 antagonist BCTC on lipopolysaccharide (LPS)-induced nitric
oxide release. BV-2 cells were treated for 15 min with various concentrations of BCTC (0.02, 0.5, 2 mmol/L), then incubated with different
concentrations (2, 20, 50 mmol/L) of p-aminophenol for a further 15 min period, before being stimulated with LPS. After 24 h, nitrite
concentrations in the BV-2 cell-free supernatants were measured using the Griess assay. Data from eight independent experiments are
normalized against nitrite concentration in samples stimulated in the absence of inhibitors. Nitrite concentration in these samples was
9.4 ± 2.4 mmol/L. The effect of treatments was assessed by the two-way analyses of variance (ANOVA), followed by Tukey’s post hoc test. No
##
significant differences were observed for stimulated cells incubated in the absence of BCTC (ANOVA F = 0.049, P = 0.99). P < 0.01, significantly
different from stimulated cells incubated in the absence of p-aminophenol (ANOVA F = 29.65, P < 0.0001). ANOVA interaction F = 0.060,
P = 0.99. BCTC: 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide

150

Nitrite (% control) 100

50

0
0 2 20 50
AM404 (µmol/L)

0 µmol/L BCTC 0.5 µmol/L BCTC
0.02 µmol/L BCTC 2 µmol/L BCTC

Figure 11. Effects of AM404 alone or in combination with the TRPV1 antagonist BCTC on lipopolysaccharide (LPS)-induced nitric
oxide release. BV-2 cells were treated for 15 min with various concentrations of BCTC (0.02, 0.5, 2 mmol/L), then incubated with
different concentrations (2, 20, 50 mmol/L) of AM404 for a further 15 min period, before being stimulated with LPS. After 24 h, nitrite
concentrations in the BV-2 cell-free supernatants were measured using the Griess assay. Data from five independent experiments are
normalized against nitrite concentration in samples stimulated in the absence of inhibitors. Nitrite concentration in these samples was
16.5 ± 2.7 mmol/L. The effect of treatments was assessed by the two-way analyses of variance (ANOVA), followed by Tukey’s post hoc test.
#
No significant differences were observed for stimulated cells incubated in the absence of BCTC (ANOVA F = 0.096, P = 0.96). P < 0.05
##
and P < 0.01, significantly different from stimulated cells incubated in the absence of AM404 (ANOVA F = 16.45, P < 0.0001). ANOVA
interaction F = 0.047, P = 0.99. BCTC: 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide
p-aminophenol and AM404 attenuated NO secretion from activated BV-2 cells in the presence of BCTC
at all concentrations used. BCTC on its own did not have a significant effect on the NO secretion by
stimulated BV-2 microglia [Figures 10 and 11] or their viability (data not shown).