SUPPLEMENTARY INFORMATION

Transkript

1 SUPPLEMENTARY INFORMATION IKK phosphorylation regulates RPS3 nuclear translocation and NF- B function during Escherichia coli O157:H7 infection Fengyi Wan 1,2, Amanda Weaver 1, Xiaofei Gao 3, Michael Bern 1, Philip R. Hardwidge 3 & Michael J. Lenardo 1 * Supplementary Fig. 1 - Fig. 13

2 Supplementary Fig. 1 Knockdown of IKK and IKK in Jurkat cells. Immunoblot for IKK, IKK, I B and -actin in Jurkat cells transfected with scrambled nonspecific (NS), IKK, or IKK sirnas. The data represent four independent experiments. DAPI Flag RPS3 Flag-IKK Flag-IKK SSEE SSAA SSEE SSAA Supplementary Fig. 2 Representative immunofluorescence images of Jurkat cells overexpressing constructive active (SSEE) or kinase-dead (SSAA) IKK construct. Jurkat cells overexpressing indicated Flag-IKK constructs were analyzed by confocal microscopy following fixation and staining for RPS3, Flag, and nuclei. Representative Flag positive (top) and Flag negative (bottom) cells from at least 200 cells in two independent experiments were shown, respectively.

3 a b c Supplementary Fig. 3 Sodium pervanadate (Pv) treatment induces I B degradation, but not nuclear translocation of RPS3 in Jurkat cells. (a) Jurkat cells were treated with indicated concentration of Pv for 2 h, and whole-cell lysates were immunoblotted as indicated. -actin was used as a loading control. (b) Jurkat cells were treated with or without 800 µm sodium pervanadate (Pv) for 2 h, and wholecell lysates were immunoblotted directly or after immunoprecipitation (IP) with anti-rps3 antibody for importin- (imp- ), RPS3 and I B. (c) Immunoblotting of cytosolic (Cyto) and nuclear (Nuc) subcellular fractions derived from Jurkat cells treated with or without 800 µm sodium pervanadate (Pv) for 2 h. Hsp90 and PARP served as the cytosolic and nuclear markers and loading controls, respectively. The data represent at least two independent experiments. Supplementary Fig. 4 The recombinant IKK phosphorylates I B (1-54) in vitro. Autoradiograph (top) and Coomassie blue-stained gel (bottom) of in vitro kinase assays performed with recombinant GST or GST-I B (1-54) protein using recombinant human IKK as kinase. Filled and open symbols indicate autophosphorylated IKK and phosphorylated I B, respectively. The data represent three independent experiments.

4 Supplementary Fig. 5 Serine 209 of RPS3 is conserved in many species. Alignment of the C-terminal tail region of RPS3 from different species. The serine 209 residue conserved in many species is indicated in red. Supplementary Fig. 6 No CK2 contaminant in the recombinant IKK proteins. IKK or IKK recombinant proteins (1 µg each), and 15 µg of whole-cell lysate from HEK 293T cells (293WCL) were separated by SDS-PAGE, followed by immunoblotted with antibodies specific for IKK / or CK2. The data represent two independent experiments.

5 5 _ Luciferase activity (fold) _ 0 pcdna pcdna-ikk Supplementary Fig. 7 Activation of NF- B by IKK overexpression. NF- B luciferase assay (mean and s.d., n=3) using Jurkat cells transfected with empty vector pcdna or pcdna- IKK plasmids together with a 5 Ig B sites-driven luciferase reporter gene. Representative results from at least four independent experiments were shown. Flag-RPS3 Flag-RPS3 (S209A) SSC GFP Supplementary Fig. 8 The S209A mutation of RPS3 does not impair translation. Jurkat cells, transfected with sirna specifically targeting the 3 untranslated region of RPS3 mrna for 48 h, were transfected again with the same sirna plus wild-type or S209A mutant Flag-RPS3 constructs together with pegfp plasmid. 24 h later, GFP expression was assessed by flow cytometer. Numbers indicate the percentage of GFP-positive cells, gated on living cells. Representative plots from three independent experiments were shown.

6 Supplementary Fig. 9 The S209A mutation of RPS3 does not attenuate the recruitment of endogenous p65 to the RPS3-independent CD25 B promoter. Jurkat cells transfected with RPS3-3 UTR sirna and Flag-tagged wild-type (WT) or S209A mutant RPS3 constructs were left untreated or stimulated with 50 ng/ml PMA plus 1.5 µm ionomycin (PMA+I) for 2 h. The cell extracts were analyzed by chromatin immunoprecipitation assays of the recruitment of endogenous p65 to the B region of CD25 promoter. p65-bound DNA was analyzed by quantitative real-time PCR (primers, above diagrams) and normalized to the input DNA, and the cells transfected with Flag-tagged WT RPS3 construct and untreated (mean and s.d., n=3). Data are from one representative of two independent experiments. * P < 0.05, Student s t-test. Supplementary Fig. 10 The S209A mutation of RPS3 attenuates the T cell receptor engagement-induced IL-8 expression in Jurkat cells. Jurkat cells transfected with RPS3-3 UTR sirna and Flag-tagged wild-type (WT) or S209A mutant RPS3 constructs were left unstimulated (Unstim) or stimulated with 1 µg/ml of anti-cd3/cd28, or 50 ng/ml PMA plus 1.5 µm ionomycin (PMA+I) for 24 h. The IL-8 in the supernatant was measured by ELISA (mean and s.d., n=3). ND, not detected. The data represent two independent experiments.

7 Supplementary Fig. 11 The S209A mutation of RPS3 does not attenuate the T cell receptor engagement-induced CD25 expression in Jurkat cells. Jurkat cells transfected with RPS3-3 UTR sirna and Flag-tagged wild-type (WT) or S209A mutant RPS3 constructs were left unstimulated (Unstim) or stimulated with 1 µg/ml of anti-cd3/cd28, or 50 ng/ml PMA plus 1.5 µm ionomycin (PMA/I) for 12 h. The induced CD25 expression on the cell surface was stained with PE-conjugated anti-cd25 antibody and analyzed by flow cytometer. The percentage of viable cells with CD25 positive were shown (mean and s.d., n=3). Supplementary Fig. 12 A subset of certain NF- B target gene transcription is not impaired by NleH1. HeLa cells were left uninfected (Uninf) or infected for 3 h with wildtype (WT) E. coli O157:H7 or stains isogenic deletions in the escn ( escn) or nleh1 ( nleh1) genes. Transcript abundance of TNFSF13B, IFNGR2, and CD25 relative to uninfected cells assessed by RT-PCR analysis. The relative mrna abundance were normalized to GAPDH expression (mean and s.d., n=3). The data represent two independent experiments.

8 Supplementary Fig. 13 NleH1 does not phosphorylate IKK in vitro. HEK 293T cells were transfected with IKK (K44A)-Flag construct. In 24 h, the cells were lysed and followed by an immunoprecipitation with anti-flag or isotype control (iso) antibodies. The immunoprecipitants were used as substrate in in vitro kinase assays with either wild-type or kinase dead (K159A) NleH1 proteins as kinases. The proteins were separated by SDS-PAGE and followed by autoradiograph (top). The expression and immunoprecipitated IKK (K44A)- Flag proteins were immunoblotted with anti-flag antibody (bottom). The data represent two independent experiments.