Initiation and termination of NF-B signaling by the intracellular protozoan parasite Toxoplasma gondii

JCS02428 Supplementary Material

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• Supplemental Figure 1 - Fig. S1. T. gondii infected cells fail to upregulate NF-kB dependent gene expression in response to TNF-a stimulation despite and nuclear accumulation of NF-kB. (A) Clonal 3T3 cells expressing GFP driven by a promoter containing NF-kB binding sites were used to assess NF-kB responsiveness of T. gondii infected cells. Cells were either left untreated, stimulated with TNF-a (10ng/ml) for 18 hours, infected with p30-RFP-expressing Rh parasites for 18 hours, or infected for 2 hours followed by 18 hour stimulation with TNF-a (10 ng/ml) – for TNF-a stimulations cells were treated with TNF-a for 1 hour, washed and then cultured for 18 hours. Similar results were observed with continual overnight stimulation. Exp.ression of GFP and RFP was assessed by flow cytometry using BD FACS Calibur and analyzed using Cell Quest (Becton Dickinson, Mountain View, CA). Histograms represent GFP expression in the popuplations of cells indicated (B) Indirect immunofluorescence analysis of T. gondii infected HFF cells treated with TNF-a. Cells were infected for 2 hours, followed by 2 hour stimulation with TNF-a (10ng/ml). Anti-p65 (green) staining reveals cytoplasmic localization in uninfected cells and nuclear accumulation in cells stimulated with TNF-a for 2 hours. ‘DAPI’ indicates staining of host cell and parasite nuclei (blue). Yellow triangles indicate parasite-containing vacuoles.

• Supplemental Figure 2A
• Supplemental Figure 2B - Fig. S2. Infection with T. gondii does not result in the nuclear accumulation of NF-kB. (A) Densitometric analysis and western staining of nuclear and cytoplasmic fractions form treated cultures. Images were analyzed using ImageJ software downloaded from http://rsb.info.nih.gov/ij/. Arbitrary intensity values for p65/RelA (red) or DAPI (blue), are shown relative to the reference line used for analysis (green). Westerns were performed on cytoplasmic and nuclear fractions from cultures treated under the same conditions indicated for immunoflorescence images. (B) Low magnification indirect immunoflorescence analysis of T. gondii infected HFF cells illustrate that the results shown in ‘A’ are not restricted to a subset of cells. Anti-p65 (red) staining reveals cytoplasmic localization in uninfected cells and nuclear accumulation in cells stimulated with TNF-a for 1 hour. ‘DAPI’ indicates staining of host cell and parasite nuclei (blue).

• Supplemental Figure 3 - Fig. S3. Low magnification indirect immunoflorescence analysis of T. gondii infected 3T3 cells doubly deficient in IkBb and IkBe demonstrates lack of nuclear accumulation of NF-kB. Anti-p65 (red) staining reveals cytoplasmic localization in uninfected cells and nuclear accumulation in cells stimulated with TNF-a for 1 hour. ‘DAPI’ indicates staining of host cell and parasite nuclei (blue).

• Supplemental Figure 4 - Fig. S4. EMSA gel from Fig. 5. To demonstrate equal labeling of double stranded oligoes as well as the appearance of a gel shift, the complete gel, containing unbound probe, is shown.