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1 Supplementary Materials for A Genome-Wide sirna Screen Reveals Positive and Negative Regulators of the NOD2 and NF-κB Signaling Pathways Neil Warner, Aaron Burberry, Luigi Franchi, Yun-Gi Kim, Christine McDonald, Maureen A. Sartor, Gabriel Núñez* *To whom correspondence should be addressed. bclx@umich.edu Published 15 January 2013, Sci. Signal. 6, rs3 (2013) DOI: /scisignal This PDF file includes: Fig. S1. Characterization of the NOD2 reporter cell line used in the genome-wide sirna screen. Fig. S2. Layout of the assay plate for the genome-wide sirna screen. Fig. S3. Data normalization. Fig. S4. Data distribution. Fig. S5. Screening assay reproducibility. Fig. S6. Screening assay robustness. Fig. S7. MDP-induced NF-κB luminescence weakly correlates with cell viability. Fig. S8. Cell viability analysis helps reduce false positives. Fig. S9. Mass spectra of WBP11 and FUS peptides isolated in a biochemical screen for NOD2-interacting proteins. Fig. S10. NCF1 is a positive regulator of NF-κB signaling. Fig. S11. ZDHHC2 is a general regulator of NF-κB signaling. Other Supplementary Material for this manuscript includes the following: (available at Table S1 (Microsoft Excel format). Primary screen results. Table S2 (Microsoft Excel format). Published NOD2-interacting proteins. Table S3 (Microsoft Excel format). ConceptGen enrichment of top 700 positive regulators. Table S4 (Microsoft Excel format). Hits supported by microarray studies. Table S5 (Microsoft Excel format). Hits supported by proteomic studies. Table S6 (Microsoft Excel format). Hits supported in the literature. Table S7 (Microsoft Excel format). References for Crohn s disease GWAS hits. Table S8 (Microsoft Excel format). 313 secondary validation hits list annotation.

2 Table S9 (Microsoft Excel format). 313 secondary validation results.

3 Fig. S1. Characterization of the NOD2 reporter cell line used in the genome-wide sirna screen. A reporter cell line derived from HEK 293 cells was engineered to stably express human NOD2 and a luciferase reporter gene downstream of tandem copies of a consensus NF-κB binding site. (A) MDP-induced NOD2 signaling was dependent on RIPK2 as assessed by NF-κB luciferase assays (top) and measurement of IL-8 by ELISA (bottom). (B) sirna-mediated depletion of RIPK2 was performed with a pool of four distinct sigenome sirna reagents targeting RIPK2 at a concentration of 20 nm. Western blotting (WB) analysis performed on both RIPK2 immunoprecipitated (IP) samples (top) and whole cell lysates (WCL, middle) was performed with anti-ripk2 antibody. Actin was used as a loading control (bottom). (C) MDP-dependent NF-κB activation was dose-dependent and specific, because no NF-κB activity was observed in response to LL-MDP, an inactive stereoisomer of MDP. (D) Stimulation of NF-κB activity in the reporter cell line by TNF-α was independent of RIPK2, but dependent on RIPK1, whereas MDPdependent stimulation was independent of RIPK1. (E) The reporter cell line also exhibited MDPand NOD2-dependent activation of p38 MAPK. Western blotting experiments were performed in duplicate, with one representative image shown.

4 Fig. S2. Layout of the assay plate for the genome-wide sirna screen. We used 384-well assay plates for the (A) primary and (B) secondary validation screens. Outer wells were left empty to minimize edge effects as a result of sample evaporation. The positions of various control sirna pools used on every assay plate are indicated.

5 Fig. S3. Data normalization. (A and B) Raw bioluminescence data (NF-κB luciferase assay, Steady Glo, Promega, upper panel) and fluorescence data (cell viability assay, Cell Titer Fluor, Promega, lower panel) were collected for each barcoded assay plate with an automated plate reader over a two month period. The sirna library was spread over 68 plates, with each plate assayed in triplicate to give a total of 204 plates. (A) The average raw values for the positive (red) and negative (black) controls on each plate are shown. (B) The positive controls were set to 100% and the negative controls were set to 0% to normalize values to enable sample comparison across assay plates.

6 Fig. S4. Data distribution. (A and B) Histogram analysis of the (A) normalized average luminescence and (B) viability readings (n = 3 experiments) for each of the 18,110 genes assayed were binned, revealing a normal distribution of data from the genome-wide screen. Most of the genes had only mild effects on (A) NF-κB luciferase and (B) viability relative to those of the NT control sirna, whereas a relatively small number of genes were at the tail ends of the distribution.

7 Fig. S5. Screening assay reproducibility. (A and B) Linear correlation analysis comparing the three replica data sets in a pairwise fashion [that is Replicate 1 vs. Replicate 2 (top), Replicate 1 vs. Replicate 3 (middle), and Replicate 2 vs. Replicate 3 (bottom)] for both the (A) NF-κB luciferase and (B) cell viability assays (n = genes) illustrates overall reproducibility. The line of best fit and linear coefficient (R 2 ) value is shown in the upper right quadrant of each graph. (C and D) Linear correlation analysis was also performed across replica assay plates (68 plates, each done in triplicate). A summary histogram of the 204 R 2 values across all replica plates is shown for (C) the NF-κB luciferase assays and (D) the cell viability assays.

8 Fig. S6. Screening assay robustness. Z-score analysis (13), with positive and negative controls on each assay plate, was performed for all 204 assay plates for both the NF-κB luciferase (dark) and cell viability assays (light). The Z-scores were nearly all > 0.5, which is generally considered acceptable for high-throughput screening.

9 Fig. S7. MDP-induced NF-κB luminescence weakly correlates with cell viability. A weak correlation between average relative normalized viability and average relative normalized luminescence (where NT sirna represents 100% viability and RIPK2 sirna represents 100% luminescence reduction) was revealed when values for all 18,110 genes were plotted. The line of best fit and linear correlation (R 2 ) coefficient is shown. Genes with a particularly low cell viability tended to have reduced NF-κB luminescence, which was likely a result of a lack of cells to produce sufficient luciferase signal. This indicates a need to correct for genes whose silencing resulted in extremely low viability.

10 Fig. S8. Cell viability analysis helps reduce false positives. (A) STRING-based (22) analysis reveals multiple connections between genes that had an average relative normalized viability of less than 60%. Only high-confidence interactions (confidence threshold >0.7) are depicted. Three highly connected sub-networks involving components of core housekeeping complexes, such as the ribosome (translation), RNA polymerase II (transcription), and cell cycle progression (the kinetochore complex and the anaphase-promoting complex) are highlighted. (B) The knockdown of the serine and threonine kinase PLK1 and its substrate WEE1 as well as of several other genes that are required for cell survival [that is, BIRC5/Survivin (63), CDC20 (64), CHEK1 (65), RPL6 (66), RRM2 (67), and SGOL1 (68)] significantly reduced NF-κB luciferase (upper panel) and cell viability (lower panel) (P <0.001). These genes would have been identified as stimulators of NF-κB signaling without accounting for reduced cell viability.

11 Fig. S9. Mass spectra of WBP11 and FUS peptides isolated in a biochemical screen for NOD2- interacting proteins. (A) A data table summarizing the international protein identification (IPI) number of the candidate, the number of independent spectra analyzed, the number of unique peptides identified, the peptide sequence, and the confidence of identification. Representative mass spectra are shown for (B) WBP11 (C) and FUS. Raw data files were converted into mzxml files, and peptides were assigned to MS/MS spectra with a SEQUEST search against the Human IPI database version The following search parameters were selected: 3 dalton precursor mass tolerance, average mass, semi-tryptic search with two or fewer missed cleavages, and oxidized methionine as a variable modification. PeptideProphet ( was used to validate peptide assignments and to perform protein inference.

12 Fig. S10. NCF1 is a positive regulator of NF-κB signaling. (A) An SiGENOME sirna pool targeting NCF1 from our primary screen reduced NOD2-dependent, MDP-induced NF-κB luciferase activity compared to a non-targeting sirna control. (B) Secondary validation experiments with ON-TARGETplus sirna pools were used to silence the indicated genes, and NF-κB activation (top) or IL-8 secretion (bottom) were tested after stimulation with MDP, E. faecalis, or S. aureus. (*P <0.05). Primary bone marrow derived macrophages cultured from WT or Ncf1-deficient mice were stimulated with MDP for 30 or 60 min. (C and D) Western blotting analysis was used to monitor NOD2 pathway activation with specific antibodies against (C) IκB and piκb or (D) p38 and phosphorylated p38. Representative Western blots are shown and pooled densitometry data were used to quantify the relative protein abundances from two independent experiments normalized to the abundance of p38 protein, which was used as a loading control. The amount of IκB in WT cells was significantly lower than that in Ncf1 / cells 30 min after stimulation with MDP, as determined by the student s t-test. No statistically significant difference was observed in the extent of p38 phosphorylation between WT and Ncf1 / cells.

13 Fig. S11. ZDHHC2 is a general regulator of NF-κB signaling. (A) Epistasis analysis was performed with sigenome sirna targeting RIPK2 and ZDHHC2 coupled with NF-κB activating constructs as described in the Materials and Methods. Knockdown of ZDHHC2 reduced NF-κB induced luciferase activity stimulated by all of the constructs tested. In secondary validation experiments, ON-TARGETplus sirna pools were used to silence the indicated genes, and NF-κB activation (top) and IL-8 secretion (bottom) were tested after stimulation with (B) MDP, (C) TNF-α, (D) E. faecalis, or (E) S. aureus. (*P <0.05).

14 Key to Supplementary Tables: Column headings Term Description GeneID NCBI Entrez Gene Identification Number Gene Symbol Gene Symbol Assigned by Thermo Scientific Gene Name Official Full Name SiG sigenome sirna OnT ON-TARGETplus sirna MDP Muramyl Dipeptide Treated TNF Human Tumor Necrosis Factor Alpha Treated Viab Fluorescent Based Viability MDP Luc Bioluminescent Based NF-κB Activation TNF Luc Bioluminescent Based NF-κB Activation MDP IL8 ELISA Based IL-8 in Supernatant after MDP stimulation TNF IL8 ELISA Based IL-8 in Supernatant after TNFα stimulation.1 Replicate1.2 Replicate2.3 Replicate3 AVG Average Relative Value of replicates