4. Th1-related gene expression in infected versus mock-infected controls from Fig. 2 with gene annotation.

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Percival Sparks
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1 List of supplemental information 1. Graph of mouse weight loss during course of infection- Line graphs showing mouse weight data during course of infection days 1 to 10 post-infections (p.i.). 2. Graph of viral titers isolate from mouse lungs- Line graphs showing the titers of virus recovered from lung tissue expressed as the mean egg infectious dose (EID 50 ) per ml of clarified homogenate on days 1, 3, and 5 p.i.. The lower limit of detection for the assay was EID 50 /ml and this is indicated on the graph as a dashed line. 3. Overall gene expression profiles in replicate and pooled infections- Hierarchical clustering diagram showing the expression profiles of the individual replicates (3x N=4) and Resolver analysis software (in silico) pooled (1x N=12) array experiments. Individual animals are denoted as numbers 1 to 3 and the in silico pooled samples are denoted with the letter P. Genes displayed were changed >2-fold (P<0.01) in at least 3 of the individual (N=4) experiments compared to uninfected controls. 4. Th1-related gene expression in infected versus mock-infected controls from Fig. 2 with gene annotation. 5. NK cell-related gene expression versus mock-infected controls from Fig. 2 with gene annotation. 6. Macrophage-related gene expression versus mock-infected controls from Fig. 2 with gene annotation. 7. Neutrophil- related gene expression versus mock-infected controls from Fig. 2 with gene annotation. 8. Cytokine and chemokine-related gene expression- Hierarchical clustering diagram showing the expression profiles of the in silico pooled (1x N=12) array experiments. Genes displayed were changed >2-fold (P<0.01) compared to mock-infected controls in at least 1 experiment. Initial list of functional genes were derived from literature. 9. Interferon-related gene expression- Hierarchical clustering diagram showing the expression profiles of the in silico pooled (1x N=12) array experiments. Genes displayed were changed >2-fold (P<0.01) compared to mock-infected controls in at least 1 experiment. Initial list of functional genes were derived from literature. 10. Dendritic cell and antigen presentation- related gene expression- Hierarchical clustering diagram showing the expression profiles of the in silico pooled (1x N=12) array experiments. Genes displayed were changed >2-fold (P<0.01) compared to mock-infected controls in at least 1 experiment. Initial list of functional genes were derived from literature.

2 List of supplemental information continued 11. Apoptosis-related gene expression- Hierarchical clustering diagram showing the expression profiles of the in silico pooled (1x N=12) array experiments. Genes displayed were changed >2-fold (P<0.01) compared to mock-infected controls in at least 1 experiment. Initial list of functional genes were derived from literature. 12. Th2 cell-related gene expression- Hierarchical clustering diagram showing the expression profiles of the in silico pooled (1x N=12) array experiments. Genes displayed were changed >2-fold (P<0.01) compared to mock-infected controls in at least 1 experiment. Initial list of functional genes were derived from literature. 13. Nitric oxide and reactive oxygen response gene expression- Hierarchical clustering diagram showing the expression profiles of the in silico pooled (1x N=12) array experiments. Genes displayed were changed >2-fold (P<0.01) compared to mockinfected controls in at least 1 experiment. Initial list of functional genes were derived from literature. 14. Bar graph showing results of quantitative real-time PCR (Taqman; ABI) analysis of selected immune response-related genes. Reactions were performed on equal mass pools of total lung RNA from the three individual biological replicate infections. 15. Bar graph showing expression of key cytokine proteins. ELISAs were performed on clarified cell lysates from lungs isolated from three replicate infected animals for TNF- (assay sensitivity 5.1 pg/ml), Ccl3 (MIP-1, assay sensitivity 1.5 pg/ml), Cxcl2 (MIP-2, assay sensitivity 1.5 pg/ml), and Cxcl10 (IP-10, assay sensitivity 2.2 pg/ml), as described in Methods section. 16. Table showing expression values of key cytokine proteins during r1918 and 2: infections. ELISAs were performed on clarified cell lysates from lungs isolated from three replicate infected animals for TNF- (assay sensitivity 5.1 pg/ml), Ccl3 (MIP- 1, assay sensitivity 1.5 pg/ml), Cxcl2 (MIP-2, assay sensitivity 1.5 pg/ml), and Cxcl10 (IP-10, assay sensitivity 2.2 pg/ml), as described in Methods section. Asterisks indicate measurements that meet a 90% probability based on a twosample unequal variance t-test. 17. Expression of genes shown in the network diagram from Fig. 4. Hierarchical clustering diagram showing the expression of genes in Fig. 4. Genes displayed were induced >2-fold (P<0.01) compared to mock-infected controls only in 1918 infected mouse lung relative to mock controls.

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Supporting Information

Supporting Information Aldridge et al. 10.1073/pnas.0900655106 Fig. S1. Flow diagram of sublethal (a) and lethal (b) influenza virus infections. (a) Infection of lung epithelial cells by influenza virus