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Figure S1 mRNA expression of MKP-1, CXCL8 and CCL5 in bronchial biopsies from mild atopic asthmatics taking inhaled budesonide. Mild asthmatics were entered into a cross-over study that has been described previously (Kelly et al., 2010). Patients were initially subjected to a 3 week washout period prior to an inhaled challenge with diluent alone (saline). Following this, patients were subjected to a further 3 week washout period prior to treatment with placebo (Plac) or drugs for 11 consecutive days with a cross-over design. Budesonide (Bud) was inhaled using a Pulmicort Turbuhaler (budesonide, 200 μg) with 2 inhalations taken twice daily and placebo was administered in a similar manner. On day 9, patients were subjected to allergen inhalation (All). Bronchoscopy was performed on day 10 (24 h after the allergen challenge). Biopsies were analysed for mRNA expression of GAPDH, MKP-1, CXCL8 and CCL5 using real-time RT-PCR. Data were obtained from matched samples from 9 individuals and are plotted as a ratio of the gene of interest/GAPDH in arbitrary units as means ± SE. Significance between Plac + All and Bud + All was tested by Mann–Whitney test. *P < 0.05.

Figure S2 GILZ protein expression is induced by corticosteroids in A549 pulmonary cells. (A) As described in Figure 2B of the main manuscript, A549 cells were treated with combinations of IL-1β (1 ng/mL) and dexamethasone (1 μM) (Dex) or were not treated. Cells were harvested at the times indicated for total protein and subsequent Western blot analysis of GILZ and GAPDH. Following densitometric analysis, data (n = 7), as a ratio of GILZ/GAPDH, were expressed as fold over untreated at t = 1 and are plotted as mean ± S.E. Significance relative to untreated was tested at each time point by non-parametric ANOVA (Friedman's) with a Dunn's post-test. (B) As described in Figure 2F of the main manuscript, A549 cells were either non-stimulated (NS) or treated with various concentrations of budesonide (as indicated) or dexamethasone (1 μM) for 6 h prior to harvesting for total protein. Western blotting was performed for GILZ and GAPDH. Following densitometric analysis, data (n = 7), as a ratio of GILZ/GAPDH, were expressed as fold over NS and are plotted as mean ± S.E. An EC50 of 3.8 × 10-9 M was obtained for the induction of GILZ protein by budesonide. Significance relative to NS was tested by non-parametric ANOVA (Friedman's) with a Dunn's post-test. Significance is indicated where: *P < 0.05, **P < 0.01 or ***P < 0.001.

Figure S3 GILZ expression is induced by budesonide in primary human airway smooth muscle (ASM) cells. (A) As described in Figure 3B of the main manuscript, primary human ASM cells were treated with budesonide (0.1 μM) and harvested at the times indicated for Western blot analysis of GILZ and GAPDH. Following densitometric analysis, data (n = 6), as a ratio of GILZ/GAPDH, were expressed as fold over untreated at 1 h and are plotted as mean ± SE. Significance relative to untreated was tested at each time point by non-parametric paired t-test. (B) ASM cells were treated with various concentrations of budesonide as indicated for 6 h prior to harvesting for total protein. Western blotting was performed for GILZ and GAPDH. Following densitometric analysis, data (n = 10), as a ratio of GILZ/GAPDH are expressed as fold over NS and are plotted as mean ± SE. An EC50 of 2.1 × 10−10 M was obtained for the induction of GILZ protein by budesonide. Significance relative to NS was tested by non-parametric ANOVA (Friedman's) with a Dunn's post-test. Significance is indicated where: *P < 0.05 or **P < 0.01.

Figure S4 siRNA-mediated knockdown of dexamethasone-induced GILZ. (A) A549 cells were incubated with control or GILZ-specific siRNA for 24 h prior to stimulation with dexamethasone (0.1 μM) for 6 h. Cell lysates were then subjected to Western blot analysis for GILZ and GAPDH. (B,C) In parallel experiments, performed at the same time as those in (A), A549 cells plated into 4-chamber slides were incubated in the presence of either control siRNA or GILZ-targeting siRNA for 24 h, prior to treatment with dexamethasone (0.1 μM). Cells were subjected to immunohistochemical analysis for GILZ. (B) Depicts representative images of untreated and dexamethasone-treated cells. (C) Representative images of dexamethasone-treated cells in the presence of either control siRNA or GILZ-targeting siRNA are shown. Magnification was either 20× or 60 × 10 as indicated.

Figure S5 (A) A549 cells were either not transfected or transfected with control plasmid (pGL3) or GILZ expressing plasmid for 24 h. Untransfected cells were also treated, or not, for 6 h with dexamethasone (0.1 μM). Cell lysates were then subject to Western blot analysis for GILZ and GAPDH. In each case, blots representative of two such experiments are shown. (B) Cells treated as in (A) were analysed in parallel for GILZ expression by immunohistochemistry. Representative images showing GILZ expression in the presence of (i) control plasmid or (ii) GILZ expressing plasmid are shown.

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