Figure legends
Figure 1. Poly (I:C) stimulates CCL5 production in bronchial
epithelial
cells.
(A) NHBE cells were stimulated with poly (I:C) for 24h, and CCL5 levels
were measured in the culture supernatant. (B–D) BEAS-2B cells were
stimulated with poly (I:C) or CpG-ODN as indicated for 24 hours (B, D)
or 12 hours (C), and the CCL5 concentration in the culture supernatant
(B, D) and CCL5 mRNA expression (C) were evaluated.
(E–G) BEAS-2B cells were stimulated with poly (I:C), IL-13, and IL-4,
as indicated, for 24 hours (E, G) or 12 hours (F), and the CCL5
concentration in the culture supernatant (E, G) and CCL5 mRNA expression
(F) were evaluated.
*P < 0.05, **P < 0.01, as compared to
medium alone, using one-way ANOVA with post-hoc Holm-Sidak’s multiple
tests to conduct selected pairwise comparisons.
pIC, poly (I:C).
Figure 2. Signal transduction
mechanisms in poly (I:C)-induced CCL5 production in BEAS-2B
cells.
(A–D) Of the TLR3-related signals, si-TLR3 (A) and si-IRF3 (B), but
neither NF-κb inhibitor BAY117082 (C) nor si-RelA (D) inhibited poly
(I:C)-induced CCL5 production. (E–I) In type I IFN-related signals,
neutralizing anti-type I IFN antibody mixture (E), si-JAK1 (F), and
JAK1/2 inhibitor ruxolitinib (G), but neither STAT1 inhibitor
fludarabine (H) nor STAT3 inhibitor Stattic (I) attenuated poly
(I:C)-induced CCL5 production. (J, K) In alternative signals, PI3K
inhibitor LY294002 (J) but not si-Erk1/2 (K) reduced poly (I:C)-induced
CCL5 production. For all experiments, BEAS-2B cells were transfected
with si-RNAs for 2 days (A–B, D, F, K) or pre-incubated with inhibitors
for 2 hours (C, E, G–I). Afterwards, these were stimulated with poly
(I:C) (0.1 μg/ml) for 24 hours,
followed by measurement of CCL5 concentrations in the culture
supernatant (A–I)
*P < 0.05, **P < 0.01, as compared to
medium alone. We used studentt -tests (A, B, D, F, K) or one-way ANOVA with post-hoc
Holm-Sidak’s multiple tests to conduct selected pairwise comparisons of
treatments (C, E, G-I).
pIC, poly (I:C).
Figure 3. Signal transduction
mechanisms in poly (I:C) and IL-13-induced CCL5 production in BEAS-2B
cells.
(A–E) Poly (I:C) and IL-13-induced CCL5 production was not reduced with
si-STAT6 (A) but was inhibited with the PI3K inhibitor, LY294002 (5µM,
B). (C–E) si-IRF3 (C), and si-JAK1 (D). The JAK1/2 inhibitor,
ruxolitinib (10µM, E), also reduced poly (I:C) and IL-13-induced CCL5
production. BEAS-2B cells were pre-incubated with siRNA for 2 days (A,
C, D) or with inhibitors for 2 hours (B, E), followed by stimulation
with poly (I:C) (0.1 μg/ml) for 24 hours.
*P < 0.05, **P < 0.01, as compared to
medium alone. We used student t -tests (A, C, D) or one-way ANOVA
with post-hoc Holm-Sidak’s multiple tests to conduct selected pairwise
comparisons of treatments (B, E).
pIC, poly (I:C).
Figure 4. Ruxolitinib is a stronger
inhibitor than fluticasone propionate for reducing CCL5 in BEAS-2B cells
treated with poly (I:C) and IL-13.
(A-B) BEAS-2B cells were pre-incubated with ruxolitinib (A) or
fluticasone propionate (B) for 2 hours, followed by stimulation with
poly (I:C) (0.1 μg/ml). The maximal percentage (%) inhibition of
ruxolitinib and fluticasone propionate against poly (I:C)-induced CCL5
production was 73.4% and 41.7%, respectively. (C) BEAS-2B cells were
pre-incubated with medium alone (control), fluticasone (FP, 1µM),
ruxolitinib (Ruxo, 10µM), or both, followed by stimulation with poly
(I:C) with and without IL-13.
P < 0.05, **P < 0.01, as compared to
medium alone. We used one-way ANOVA with post-hoc Holm-Sidak’s multiple
tests to conduct selected pairwise comparisons of treatments.
pIC, poly (I:C).