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).