Cutrupi et al., 2011

Supplemental materials and methods

Materials: The p38 MAP kinase inhibitor SB203580 and the MEKK1 inhibitor U0126 were purchased from Calbiochem(San Diego, CA).

Coimmunoprecipitation: HEK293T cells were lysed with Lysis Buffer (20 mMTris-HCl pH 8.0, 20 mMNaCl,

0.5% NP-40, 1 mM DTT, 1 mM PMSF, 2 mM Na3VO4, 50 mMNaF, 1X PIC) for 10 min. Nuclei were recovered by centrifugation at 12000xg for 10 sec. The nuclear pellet was resuspended in S1 Buffer (250 mM Sucrose,

10 mM MgCl2, 1X PIC), layered on a cushion of S3 Buffer (350 mM Sucrose, 0.5 mM MgCl2, 1X PIC) and centrifuged at 8000xg for 10 min. The clean pelleted nuclei were then resuspended in Nuclear Lysis Buffer

(20 mMHepes pH 7.9, 25% glycerol, 420 mMNaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.1% NP-40, 1 mM PMSF, 2 mM Na3VO4, 50 mMNaF, 1X PIC), put on ice for 30 min and centrifuged at 12000xg for 2 min. The supernatant nuclear extracts were diluted in CoIP Buffer (20 mMHepes pH 7.9, 10% glycerol, 5 mM

MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.1% NP-40, 1 mM PMSF, 2 mM Na3VO4, 50 mMNaF, 1X PIC). 1 µg of anti- Flag antibody or a pool of 3 µg of anti-Tab2 K-20, 3 µg of anti-Tab2 E-20 and 3 µg of anti-Tab2 H-300 antibodies were used. The samples were incubated overnight at 4°C, then 50 μL of protein G sepharose beads in immunoprecipitation buffer were added, and incubation continued for another 2 hours. The pellet was washed 4 times in immunoprecipitation buffer. The samples were analyzed by immunoblotting with the appropriate antibodies.

In vitro pull-down: Purification of MBP-fusion proteins (MBP-Tab2full-lenght, MBP-406-531 Tab2) were performed according to manufacturers’ protocol (New England Biolabs, Ipswich, MA, USA). HEK293T cell lysate was prepared by adding 300 μl of Lysis Buffer 1X ( 150 mMNaCl, 2 mMTris/HCl pH 7.8, 2 mM EDTA, 0.5% NP-40, 2 mM Na3VO4, 1X PIC, 1 mM PMSF). MBP-Tab2 fusion constructs (10 μg) were preincubatedin pull-down buffer (125 mMNaCl, 20 mMTris/HCl pH 7.8, 10% glycerol, 0.1% NP-40, 0.5 mM DTT, 1X PIC, 1 mM PMSF) with 10 μM each ERα or ERα Mutfor 1 hour on rotation at 4°Cand then mixed with HEK293T cell lysate for 1.5 hours at 4°C under rotation.A volume of 50 μl of prewashed Amylose Resin beads wasthen addedfor 1 hour at 4°C on rotation. The pro- tein complexes were washed 3 times with pull-down buffer, collected by centrifugation at 12000xg, resus- pended in SDS-gel loading buffer and analyzed by immunoblotting with the appropriate antibodies.

RNA labeling, microarray hybridization and analysis: totRNA quality, concentration and labeling were checked by means of RNA 6000 nano chip assays (Agilent Technologies) and NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Waltham, MA). Only samples with RNA integrity number greater 1 Cutrupi et al., 2011 than 9 and 280/260 RNA ratio around 2 were utilized for the experiments.For each sample, mRNA was amplified starting from 1ug of totRNA by means of Amino Allyl MessageAmp II aRNA Kit (Ambion Inc.) to obtain amino allyl antisense RNA following the method developed by Eberwine and coworkers. One round of amplification was performed to obtain the necessary quantity of aaRNA for labeling using NHS ester Cy3 or Cy5 dies (Amersham Biosciences, Buckinghamshire, UK). The same quantity of differentially labeled samples was put together, fragmented and hybridized to oligonucleotide glass arrays representing 41K human unique genes and transcripts (Human Whole Genome Oligo Microarray Array 4x44K, Agilent Technologies, Palo Alto, CA). All steps were performed applying the Agilent two-color hybridization protocol. Then, slides were scanned with the dual-laser microarray scanner Agilent G2505B (Agilent Technologies). The TIFF images generated by the scanner were loaded into the Feature Extraction software (Agilent Technologies) which was used to extract raw data. Data analysis steps were done with statistical computing software “R” and packages from Bioconductor open source software for bioinformatics. In particular, the Limma package (Smyth G. K. et al., 2005) was used for preprocessing and differential expression analysis. The raw intensities were first background corrected using method“normexp” and normalized with the “loess” function. To have similar log-ratio distributions across all arrays “Aquantile” normalization was performed. To couple biological replicates and average values of replicated probes, “biolrep” and “avereps” functions were applied. Averaging the two technical replicates for each sample resulted in only one hybridization per sample. Each uRNA sample pair (siTab2/siCtrl) was labeled with Cy3 and Cy5 using the indirect method (van Gelderet al.,1990) (Amino AllylMessageAmp II aRNA Kit, Ambion Inc.).Each Tab2 siRNA treated sample was co-hybridized with the corresponding control sample treated with untargetingsiRNA. Two replicates, with dye swap, have been performed for each sample. The same quantity of differentially labeled samples was put together, fragmented and hybridized to microarray glass slides.

Statistical analysis: Statistical analysis of three-to-five replicate experiments were run on normalized values, using the nonparametric Mann-Whitney U statistics (PASW Statistics 18, SPSS, Inc., 2009, Chicago, IL, www.spss.com).

Supplemental References Van Gelder RN, von Xastrow ME, Yool A, Dement DC, Barchas JD, Eberwine JH (1990). Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci USA87:1663–1667.

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Legends to Supplementary Figures and Tables

Supplemental Table S1: The list of 282 genes corresponding to probes differentially regulated by Tab2 siRNA in TamR cells. This list contains all the genes that are significantly regulated in at least two out of three cell lines (Column 3 indicates the significant calls). Column two indicates the genes that are present in the top network (Fig.4). LogFC represents the log 2 of the ratio between siTab2 versus siCtrl samples. P- value is the probability given by the modified t-test of LIMMA.

Supplemental Table S2: Original report from Ingenuity Pathway Analysis (Ingenuity Systems, Inc) of 282 genes corresponding to probes differentially regulated by Tab2 siRNA in TamR cells (Table S1).

Supplemental Table S3: Genes regulated by estrogen in the NURSA GEMS dataset (Ochsner et al., 2009) (www.nursa.org/gems) were selected at a value of q<0.05 and overlapped to Tab2-regulated gene list (Table S1).

Supplemental Figure S1:Immunoblotting analysis of Tab2 phosphorylation in TamR cells. Cells were cultured in 1% dextran/charcoal-treated (DC) FBS supplemented with 10-6 M 4OHT for 3 days and then treated with 10 µM SB203580 or 10 µM U0126 for 8 hours. TamR cells were then subjected to cellular fractionation and the Tab2 protein analyzed by immunoblotting in the cytoplasmatic and nuclear fractions.

Supplemental Figure S2: Tab2 downregulation restores tamoxifen response in TamR cells. MCF7 wt and TamR cells were transfected with control or Tab2 siRNA and 48 hours later treated with 10-8 M E2 (white bars) or 10-8 M E2 plus 10-6 M 4OHT (black bars). After 24 hours, the proliferation was measured by two- hours bromo-deoxyuridine incorporation. Data are means ± st. dev. of replicate experiments with N=3 for TAMR-4.1, N=4 for MCF7 wt and TAMR-4.2, N=5 for TAMR-8. The date are from the same experiments presented in Figure 1. Asterisks denote “P”=0.05 (*), “P”<0.025 (**), “P”<0.01 (***); ns =nonsignificant.

Supplemental Figure S3: Canonical pathway analysis of BRCA1. The canonical pathway most significantly enriched with Tab2 regulated genes is “Role of BRCA1 in DNA Damage Response” (P < 7.7E-06). Genes significantly upregulated are indicated in red and significantly downregulated genes are indicated in green. Color intensity indicates extent of upregulation or downregu- lation.

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