neutrophils (CD11b+, the total Statistical multiple

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1 Supplementary Figure 1. (A) Wild type and Vim / mice were treated with (24mg/kg LPS); lungs were harvested at 48h and enzymaticallyy digested. CD45+ cells were excluded from the total cell population and characterized via flow cytometry as follows: alveolar macrophages (Siglec F+, CD11b, CD11c+, CD64+), neutrophils (CD11b+, Ly6G+), interstitial macrophages (CD11b+, MHC II+ +, CD11c+, CD64+, CD24 ). Data is expressed as a percentage of the total CD45+ cell population. (B) Real time RT PCR analysiss of IL 1β mrna expression in WT and Vim / mice that were challenged with a sub lethal dose of LPS for 48 hr. Data represented are means ± SD from two independent experiments, each with at least two animals per experiment. Statistical analysess were performed using one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.

2 Supplementary Figure 2. (A) WT and Vim / mice were treated with PBS containing either 200 µg of asbestos crocidolite or the control particle titanium dioxide (TiO 2 ), intratracheally. Fortyfrom eight hours later the lungs were enzymatically digested. CD45+ cells were excluded the total cell population and characterized via flow cytometry as follows: alveolar macrophages (Siglec F+, CD11b, CD11c+, CD64+), neutrophils (CD11b+, Ly6G+), interstitial macrophages (CD11b+ +, MHC II+, CD11c+, CD64+, CD24 ). Data is expressed as a percentage of the total CD45+ cell population. (B) Vim / mice are protected from asbestos induced ALI, as assessed histologically by H&E staining. Data in are from two independent experiments of n = 4 6 animals per group. Statistical analyses were performed using one way ANOVA with a correction provided by the Bonferroni multiple comparisons test. Scale bars, 200 µm.

3 Supplementary Figure 3. (A) Wild type and Vim / mice were treated with (0.07U bleomycin, 5d); lungs were harvested and enzymatically digested. CD45+ cells weree excluded from the total cell population and characterized via flow cytometry as follows: alveolar macrophages (Siglec F+, CD11b, CD11c+, CD64+), neutrophils (CD11b+, Ly6G+), interstitial macrophages (CD11b+ +, MHC II+, CD11c+, CD64+, CD24 ). Data is expressed as a percentage of the total CD45+ cell population. (B) Real time RT PCR analysiss of IL 1β mrna expression in WT and Vim / mice that were challenged with bleomycin as described above. Data represented are means ± SD from two independent experiments, each with at least two animals per experiment. Statistical analysess were performed using one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.

4 Supplementary Figure 4. (A) Quantitative analysis of Masson s Trichrome stains for collagen from WT and Vim / lungs 21 d following intratracheal instillationn of bleomycin or PBS. Images in Figure 4A and this panel represent data obtained from at least three animals per group; data shown are mean ± SD., ***P< relative to WT versus Vim /. (B) Elastic modulus frequency plot obtained from live, unfixed lung tissue of saline and bleomycin treated WT and KO animals. Microindentation data were fit using the Hertz model to acquire the elastic modulus of regions of lung tissue. Statistical analyses were performed using one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.

5 Supplementary Figure 5. Confocal micrographs of NLRP3 (green) and ASC (red) and chromatin (blue) in J774.1 macrophages (WT) and J774.1 macrophages expressing shrna against vimentin (KD). Macrophages were either activated as described in (Figure 8) or left untreated prior to fixing and staining with anti NLRP3 images are representative of at least three independent experiments. Scale (1:100), and anti ASC (1:200) antibodies and Hoechst (1 g/ml). Confocal bars, 10 m.

6 Supplementary Figure 6. Vimentin interacts with components of the NLRP3 inflammasome. (A) Vim / BMDM cell extracts were prepared either from unactivated (control) or MSU treated cells. Shown are fractions from the unbound (U), first wash (W), and bound (B) samples. (B) In activated Vim / BMDM cells, approximately 3x more NLRP3 was bound to vimentin than in control cells, n=4. (C) After priming and activation of Vim +/+ and Vim / BMDM cells with LPS and ATP, cells lacking vimentin (1:1000) do not show substantial changes in NLRP3 (1:1000), ASC (1:333), actin (1:1000), or tubulin (1:1000), n=4. (D) The ponceau S stained membrane as well as the western blots for the regions shown in (A) are included. The membrane was cut into three parts and then probed with antibodies directed against NLRP3 (1:1000), caspase 1 (1:1000), ASC (1:333) and vimentin (1:1000). The numbers next to the western blot images indicate where molecular weight standards were located on the membrane (kd). Statistical analysess were performed using one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.

7 Supplementary Figure 7. The full length western blots that weree used to prepare Figure 6C and Figure 6D are shown; (A) and (B) respectively. The numbers next to the western blot images indicate where molecular weight standards were located on the membranee (kd). All antibodies were used at a dilution of 1:1000 in 1x TBS with 0.1% Tween 20.

8 Supplementary Figure 8. The full length western blots that weree used to prepare Figure 7A are shown. The numbers next to the western blot images indicate where molecular weight standards were located on the membranee (kd). All antibodies were used at a dilution of 1:1000 in 1x TBS with 0.1% Tween 20.

9 Supplementary Figure 9. The full length western blots that weree used to prepare Figure 8A and 8B are shown; (A) and (B) respectively. The numbers next to the western blot images indicate where molecular weight standards were located on the membrane (kd). Partial lanes are shown where the membrane was cut and was used to probe for a different protein. All antibodies for western blotting were used at a dilution of 1:1000 in 1x TBS with 0.1% Tween 20.