SUPPLEMENTARY EXPEMENTAL PROCEDURES

Transcription

1 SUPPLEMENTARY EXPEMENTAL PROCEDURES Plasmids- Total RNAs were extracted from HeLaS3 cells and reverse-transcribed using Superscript III Reverse Transcriptase (Invitrogen) to obtain DNA template for the amplification of the RelA, RelB, c-rel, p50 and p52 open reading frames. For RelA, RelB, c-rel and p52, HeLaS3 cells were treated with TNF-alpha prior to DNA preparation. PCR reactions were performed using the primers listed in Table S1. All forward and the reverse primers contained EcoRI and HindIII sites, respectively. The reverse primers for p50 and p52 cdnas were designed to generate stop codons after the His433 and Asn477 residues at which these proteins are cleaved from their precursors, p105 and p100 respectively. PCR products for RelB, c-rel, p50 and p52 were purified with QIAquick Gel Extraction Kit (Qiagen), digested with EcoRI and HindIII, and inserted between the EcoRI/HindIII sites of pbluescriptii SK+ (Toyobo) to generate SK-RelB, SK-c-Rel, SK-p50 and SK-p52, respectively. The PCR fragment encoding RelA was inserted into a pgem-t Easy vector (Promega) which was then digested partially with EcoRI and fully with HindIII and inserted into the EcoRI/HindIII sites of pbluescriptii SK+ to generate SK-RelA. Finally, each plasmid was confirmed to have the same DNA sequence as that in RefSeq. RelA contained an amino acid replacement in the translated product, P180S, which is specific to HeLa S3 cells. However, the protein remains functional in this cell line. SK-RelA was digested with BamHI and HindIII and SK-RelB, SK-c-Rel, SK-p50 and SK-p52 were digested with EcoRI and HindIII to obtain cdna fragments containing the entire coding sequences of each NFκB protein. These fragments were then ligated to prk5 digested with BamHI and HindIII for RelA or with EcoRI and HindIII for other NF-κB proteins to generate RK5-RelA, RK5-RelB, RK5-c-Rel, RK5-p50 and RK5-p52, respectively. A 2.4kb SalI/NotI fragment was isolated from pcmv-sport6-req (Invitrogen), which contains the full length human requiem open reading frame (REQ; Genebank accession number bc014889) and this was inserted between the SalI-NotI sites of pbluescript ΙΙSK + to generate pbs-sk-req. Using this plasmid as the template PCR was performed using primers shown in Table S1. The resulting product was digested with XhoI and NcoI and inserted between the corresponding sites of pbs-sk-req to generate pbs-sk-req2. A 1.6kb XhoI/XbaI fragment of pbs-sk-req2 was inserted between the corresponding sites of pbs-sk to generate pbs-sk-req3. pbs-sk-req3 was first digested with XhoI, Klenow-treated and then digested with XbaI. The resultant 1.6kb fragment was inserted between EcoRI (blunt ended-by Klenow) and Xba1 site pcagf to generate pcagf-req-ig. Oligonucleotides FLAG-f and FLAG-r were inserted into the BamH1-Xho1 fragment of pmxs-iresneo (pmxs-in) to generate pmxs-flag-in. A 2.4 kb Xho1-Not1 fragment of 1

2 pbs-req2 was inserted into the Xho1-Not1 sites of pmxs-flag IN to generate pmxs-flagreq-in. A 2.4 kb XhoI/NotI fragment of pbs-req2 containing full length REQ cdna was inserted between the XhoI-NotI sites of pgex-4t-1 (GE healthcare) to generate pgex-req. After digesting pgex-req with BamHI/NotI or with StuI/NotI, the longer fragments were Klenow-treated and self-ligated to produce pgex-ct1 and -CT2, respectively. pgex-req was digested with PpuMI/NotI and ligated with a pair of nucleotides (REQ-f2, -r2; Table S1) to generate pgex-cx3. Using pbs-sk-req2 as the template and the REQ-f3, -r3 primers (Table S1), PCR was performed. The BsrBI/NotI fragment of the resultant PCR product was inserted between BsrBI/NotI sites of pgex-req to generate PGEX-CT4. pgex-req was digested with XhoI /BlpI, with XhoI/BamHI (partial digestion) or with XhoI/StuI, and the resultant longer fragments were Klenow-treated and self-ligated to generate pgex-nt1, -NT2 and NT3, respectively. PCR was performed using pbs-sk-req2 as the template and the REQ-f4 and r4, or REQ-f5 and r4 primer pairs (Table S1). XhoI/NotI fragments of the resultant PCR products were inserted between the corresponding sites of pgex-4t-1 to generate pgex-nt4 and -NT5, respectively. MMP1TRE oligonucleotide (MMP1TRE-f and r; TableS1) was annealed and inserted into BglI-KpnI site of pgl4.12 (Promega) to generate pgl-mmp1tre. 3κB luciferase vector was reported previously (1). The DNA fragment containing blasticidin resistance gene was amplified from plenti6/v5-d-topo and inserted into BamHI-XbaI site of pcs2 to generate pcs2-blasticidin. The 1kb BamHI-NotI fragment of pcs2-bla containing blasticidin resistance gene and SV40 polyadenylation signal was inserted into BamHI-NotI site of pcdna3.1 (Invitrogen) to generate pcdna3.1-blasticidin. The 1.9kb BglII-XhoI fragment of pcdna3.1-bla containing CMV promoter, blasticidin resistance gene and SV40 polyadenylation signal was inserted into BamHI-SalI site of pgl4.12 (Promega) to generate pgl-cb. The oligo nucleotide containing NFκB sites of HIV-1 and minimal promoter (κb-minipro-f and -r: Table S1) was inserted into the multi cloning site of pgl-cb to generate pgl-hivκb-mlcb. Two pairs of oligonucleotides (shreq#1-f and r, shreq#2-f and r, respectively; Table S1) were inserted between the BbsI/EcoRI sites of pmu6 after annealing to generate pmu6-shreq#1 and pmu6-shreq#2. A BamHI-EcoRI fragment of pmu6-shreq#1 and pmu6-shreq#2 were inserted between BamHI/EcoRI sites of psssp to generate psssp-shreq#1 and psssp-shreq#2, respectively. SUPPLEMENTARY FIGURE LEGENDS Supplementary Fig. S1. LC-MS analysis of FLAG-Brm- and FLAG-BRG1-2

3 immunoprecipitates revealed REQ as one of their interacting protein. A.Silverstain of FLAG-Brm- and FLAG-BRG1- immunoprecipitates. Nuclear extracts from 293T cells transfected with pcagf vector (Empty), FLAG-Brm- or FLAG-BRG1- expressing vector were immunoprecipitated with anti-flag antibody. Each immunoprecipitates were separated by SDS-PAGE and visualized using silver stain plus (Bio-Rad) under manufacturer s recommendation. B. Number of peptide hits to SWI/SNF subunits and their interactors by LC-MS analysis of FLAG-Brmand FLAG-BRG1- immunoprecipitates. C. Peptide sequences of REQ detected in FLAG-Brm- and FLAG-BRG1- immunoprecipitates by LC-MS analysis. Supplementary Fig. S2. Evaluation of the shrna-mediated knockdown efficiency. A. Effects of the knockdown of endogenous Brm, BRG1 and REQ (shreq#1) in 293FT cells by shrna. Total RNAs were extracted and the expression levels of Brm, BRG1, REQ or GAPDH (control) were determined by semi-quantitative RT-PCR analysis. B. Effects of the knockdown of endogenous Brm, BRG1 and REQ in HT-29 cells. HT-29 cells were transduced with shrna expression retrovirus vectors targeting Brm, Brg1, REQ (shreq#1) and GFP (control) and whole cell lysates were then prepared and analyzed by immunoblotting using anti-brm, anti-brg1, anti- REQ and anti-actin antibodies respectively. Arrowhead indicates non-specific band and arrow indicates the predicted molecular weight of REQ protein. C. Effects of the knockdown of endogenous REQ in 293FT cells by shreq#1 and shreq#2. Total RNAs were extracted and semi-quantitative RT-PCR analysis was performed for REQ or GAPDH (control) genes. Supplementary Fig. S3. REQ and Brm specifically promote RelB/p52-dependent transcriptional activity. Similar transfection experiments to that shown in Figure 2C the left panel are repeated twice, here 293FT cells were transfected with expression vectors for RelA/p50, RelB/p52 or empty vector and with shrnas against Brm, BRG1, REQ (#1 and #2) or GFP (control). At three days after transfection, RNA was extracted and the endogenous BLC, ELC or GAPDH gene levels were analyzed by semi-quantitative RT-PCR. Densitometric analysis of these three gels (Fig. 2C left panel and Fig. S3) were summarized after normalization as a bar graph in Figure 2C the right panel. Supplementary Fig. S4. BLC activation after lymphotoxin treatment requires either REQ or Brm in HT-29 cells. Similar lymphotoxin-treatment experiments to that shown in Figure 4A the left panel are repeated twice, here HT-29 cells were stably transduced with shbrm, shbrg1, shreq (#1 and #2) or shgfp (control) expressing retroviral vectors. After a 6 h treatment with LT (100 ng/ml) or no LT (+LT and LT, respectively), total RNAs were extracted to 3

4 determine the expression levels of the endogenous BLC and GAPDH genes by semi-quantitative RT-PCR. Densitometric analysis of these three gels (Fig. 4A left panel and Fig. S4) were summarized after normalization as a bar graph in Figure 4A the right panel. Supplementary Fig. S5. The knockdown of Brm, BRG1 and REQ does not affect the processing of p100 to yield p52. HT-29 cells stably transduced with shrna expression vectors against Brm, BRG1, REQ and GFP (control) were treated with or without lymphotoxin. After 6 hours, total cell proteins were prepared and analyzed by immunoblotting using anti-relb, anti-p100/p52 and anti-actin antibodies respectively. Supplementary Fig. S6. BLC gene activation by lymphotoxin is RelB-dependent. HT-29 cells stably transduced with shrna expression vectors against RelB and GFP (control) were treated with or without lymphotoxin. After 6 hours, total RNA were extracted to determine the expression levels of the endogenous BLC and GAPDH genes by semi-quantitative RT-PCR. SUPPLEMENTARY REFERENCES 1. Gohda, J., Irisawa, M., Tanaka, Y., Sato, S., Ohtani, K., Fujisawa, J., and Inoue, J. (2007) Biochem Biophys Res Commun 357(1),

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