Luo et al. Supplemental Figures and Materials and Methods

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1 Luo et al. Supplemental Figures and Materials and Methods The supplemental figures demonstrate that nuclear NFAT is situated at PODs, overexpressed PML does not increase NFAT nuclear localization, and PML knockdown moderately decreased nuclear contents of NFATc. The supplemental Materials and Methods describe additional materials and experimental procedures used in this study. 1

2 Luo et al. Supplemental Figure 1 Supplemental Figure 1. Co-localization of NFATc and PML in PODs. (A) Nuclear co-localization of NFATc and PML in Jurkat cells. Jurkat cells were either unstimulated or activated for 8 hours by TPA/A23187 and were then fixed, permeabilized, stained with DAPI, anti-pml, and anti-nfatc antibodies, and examined under confocal microscope. Bar indicates 10 m. The overlapping image of PML and NFATc suggests an intra-nuclear co-localization of NFATc and PML. (B) Activation of Jurkat resulted in the presence of NFATc in the nucleus. 400 cells were counted for each treatment described in (A), and percentages of cell with NFATc speckles were calculated. Results were from two experiments. (C) Quantitation of NFATc speckles in Jurkat cells. Jurkat cells with different number of NFATc speckles were calculated. (D) Quantitation of NFATc-PML co-localization. The percentage of NFATc speckles that were co-localized with PML was calculated. Results are average of two experiments. 2

3 Luo et al. Supplemental Figure 2 Supplemental Figure 2. PML-dependent nuclear presence of NFATc and NFATp. (A, B, C) PML overexpression did not increase nuclear localization of NFAT. Jurkat cells (A) and DO11.10 T cell hybridoma (B, C) transduced with PML-VI or its YFP control were stimulated with TPA/A23187 and nuclear extracts were prepared. The nuclear NFATc contents in Jurkat (A) and DO11.10 cells (B) 2 h and 4 h after activation were determined using anti-nfatc (7A6), while the amount of nuclear NFATp in DO11.10 cells (C) 20 min, 40 min, and 60 min after activation were determined by anti-nfatc (4G6-G5). The presence of PML-VI-myc in PML-transduced T cells was demonstrated by immunoblotting with anti-myc. (D, E) PML-knockdown moderately affected nuclear presence of NFAT. The nuclear contents of NFATp (D) and NFATc (E) in vector control and PMLsiR Jurkat cells stimulated with TPA/A23187 were determined as in (A-C). 3

4 Supplemental Materials and Methods Reagents and cell culture A23187, TPA, and concanavalin A (Con A) were purchased from Sigma Chemical Co. (St. Louis, MO). NFAT-CAT, containing 5 tandem repeats of murine IL-2 promoter distal NFAT site (-280 bp), has been previously described (Ho et al., 1997). Murine NFATc promoter (-752 to -21 bp) was isolated by PCR using 5 primer TGC AAT CTG TTA GTA ATT TAG CGG GA and 3 primer GTT CGG AAC CTC TCG GTC TCA, and was subcloned into the pgl2-basic luciferase reporter vector (Promega, Madison, WI). T cell were cultured in RPMI media with 10% fetal calf serum (both from GIBCO, Grand Island, NY), 10 mm glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and M 2-ME. Dulbecco s Modified Eagle s Medium (DMEM) was used instead of RPMI in the culture of 293T and 293 cells. Nuclear extracts, total cell extracts, and immunoblots were prepared and performed as previously described (Wu et al., 2003a). Retroviral infection of T cells PML-VI was sub-cloned into pgc-ires-yfp, a homologue of pgc-ires-gfp (a gift from Dr. Gina Costa, Stanford University, Stanford, CA) to generate pgc-pml-ires-yfp. Retroviruses were generated by transfection of Phoenix cells (gifts of Dr. Garry P. Nolan, Stanford University) with 10 μg of pgc-yfp or pgc-pml-yfp plasmids. Phoenix cell supernatants containing retrovirus were collected 48 h after 4

5 transfection. Viral titers were determined using NIH 3T3 cells, and virus stocks with titers greater than 1 x 10 6 were used for spin infection of DO11.10 or Jurkat-Eco T cells. 48 hours after infection, YFP-expressing Jurkat and DO11.10 cells were isolated by fluorescence sorting on FACSVantage SE (Becton Dickinson, Mountain View, CA). RT-PCR Jurkat cells (2x10 6 each), resting or activated by TPA (100 ng/ml) and ionomycin (500 ng/ml) at different time points, were harvested and lysed by TRIZOL Reagent (Gibco Invitrogen), and RNA prepared. 3 μg of total RNA was used for reverse transcription using MMLV reverse transcriptase, with -actin as an internal control. The PCR primers are: htnf -f 5 -AGC CCA TGT TGT AGC AAA CC-3, htnf -r 5 -GGA AGA CCC CTC CCA GAT AG-3 ; htrail-f 5 -GCC CAT GAA TTA CCC ATG TC-3, htrail-r 5 -TCC TTG ATG ATT CCC AGG AG-3 ; hnur77-f 5 -AGA GCT ATT CCA TGC CTA CG-3, hnur77-r 5 -GTT CGG ACA ACT TCC TTC AC-3 ; mtnf -f 5 -ACG GCA TGG ATC TCA AAG AC-3, mtnf -r 5 -CGG ACT CCG CAA AGT CTA AG-3 ; hfasl-f 5 -GGC CTG TGT CTC CTT GTG AT-3, hfal-r 5 -CAA GAT TGA CCC CGG AAG TA-3 ; hil2-f 5 -TGC AAC TCC TGT CTT GCA TT-3, hil2-r 5 -ATG GTT GCT GTC TCA TCA GC-3. Chromatin Immunoprecipitation (ChIP) and Re-ChIP Jurkat cells were cultured in complete RPMI medium with 10% fetal calf serum. 1 5

6 x 10 7 cells were used for each time point. Jurkat cells were stimulated with or without TPA (50 ng/ml)/a23187 (250 ng/ml) for 4 hours, washed, cross-linked with 1% formaldehyde at room temperature for 10 min, quenched by 125 nm glycine, and washed with ice cold PBS contained protease inhibitor cocktail. Cells were suspended in cell lysis buffer (5 mm PIPES ph8.0, 85 mm KCl, 0.5% NP-40 and protease inhibitor cocktail) (1x10 7 cells in 1 ml) and incubated on ice for 10 minutes. Nuclei were collected by centrifugation, resuspended in 300 μl nuclear lysis buffer (50 mm Tris-HCl PH8.1, 10 mm EDTA, 10% SDS, and protease inhibitor cocktail) on ice for 10 minutes. The nuclei were then sonicated on ice to disrupt nuclear DNA to an average length of about 500 bp, and debris was removed by centrifugation (14,000 rpm for 10 minutes at 4 C on an Eppendorf 5415C centrifuge). Supernatant, containing chromatin, was carefully transferred to a new tube. Fragmented chromatins were pre-cleared with protein-g-sepharose beads (blocked by PBS containing 1 mg/ml salmon sperm DNA, 1 mg/ml BSA, and protease inhibitor cocktail) at 4 C for 4 hours. Protein-G-sepharose beads were incubated in IP dilution buffer (0.01% SDS, 1.1% Triton-X 100, 1.2 mm EDTA, 16.7 mm Tris-HCl ph8.1, 167 mm NaCl, and protease inhibitor cocktail) with anti-nfatc1 antibody (7A6, BD-PharMingen), anti-pml (PG-M3, Santa Cruz), anti-cbp (A-22, Santa Cruz), or mouse IgG control (Sigma) at 4 C over night. 25 μl of antibody-loaded protein-g-sepharose beads were then added to samples and incubated for one hour. Beads were collected by centrifuge and washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton-X 100, 6

7 2 mm EDTA, 20 mm Tris-HCl ph 8.1 and 150 mm NaCl), high salt wash buffer (0.1% SDS, 1% Triton-X 100, 2 mm EDTA, 20 mm Tris-HCl ph 8.1 and 500 mm NaCl), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mm EDTA, and 10 mm Tris-HCl ph 8.0), and TE buffer. DNA-protein complexes were eluted from Sepharose beads by 300 μl elution buffer (1% SDS and 0.1 M NaHCO 3 ), 1 μl RNase A (10 mg/ml) and NaCl to a final concentration of 0.3 M were added, and the mixture incubated at 65 C for 5 hours to reverse formaldehyde cross-linking. 2.5 volumes of ethanol were added to samples, and chromatins were precipitated at -20 C overnight. Pelleted chromatins were dissolved in 100 μl TE with 5X PK buffer (50 mm Tris-HCl ph 7.5, 25 mm EDTA, 1.25% SDS) and 1.5 μl proteinase K (10 mg/ml), incubated at 45 C for 2 hours. Chromatins were then purified by Qiagen column. 5 μl of each eluted sample was used for PCR using the primers hil2p-f 5 -TAC AGA AGG CGT TAA TTG-3 and hil2p-r 5 -CAA TTA ACG CCT TCT GTA-3. In Re-ChIP experiments, complexes were eluted from Qiagen clean-up columns by incubation for 30 min at 37 C in 25 μl 10 mm DTT. After centrifugation, the supernatant was diluted 20 times with Re-ChIP buffer (1% Triton X-100, 2 mm EDTA, 150 mm NaCl, 20 mm Tris-HCl, ph 8.1) and subjected to the same procedure as described for ChIP. 7