Supplementary Materials and Methods
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Supplementary Materials and Methods Antibodies and reagents The goat polyclonal antibodies against TOX3, the rabbit polyclonal antibody against Ki-67, the mouse monoclonal antibody against BRCA1、DNMT1 and GAPDH, and the secondary goat anti-rabbit biotinylated (IgG) antibody, rabbit anti-goat HRP-conjugated antibody and goat anti-mouse HRP-conjugated antibody were purchased from Abcam (Cambridge, MA, USA). The rabbit polyclonal antibody against TOX3, the mouse monoclonal antibodies against HA and Myc epitopes were products of Sigma-Aldrich (St. Louis, MO, USA). MTT, DMSO, Albumin from bovine serum (BSA), Mayer’s hematoxylin, 3-3’-diaminobenzidine (DAB), Cisplatin (CDDP), 17-β estradiol (E2), trichostatin A (TSA), G418 and puromycin were also products of Sigma-Aldrich. cDNA, shRNA and reporter constructs The full length TNRC9 was PCR amplified using forward 5’- GGCCAATCCGGCCATGGATGTGAGGTTCTACC-3’ and reverse 5’- GGCCTCTAAGGCCTCAGAAAATACTGACCTGCGATAATACTTGAGT-3’ primers and subsequently subcloned into SfiI fragment of pCDEF3 vector (Invitrogen, Carlsbad, CA) with an N-terminal HA tag. The HA-TNRC9mut is a TNRC9 DNA binding domain- deleted truncation of HA-TNRC9 with a deletion from 922bp to 1149bp of NM_001080430. The full length CREB was PCR amplified using forward 5’- GGCCAATCCGGCCATGACCATGGAATCTGGAG-3’ and reverse 5’- GGCCTCTAAGGCCTTAATCTGATTTGTGGCAGT-3’ primers and subcloned into SfiI fragment of pCDEF3 vector with an N-terminal Myc tag. The shRNA construct targeting TNRC9 were established using pSuper RNAi system (Oligoengine, Seattle, WA). The targeting regions were 1931bp-1949bp (sh1) and 974bp-992bp (sh2) of NM_001080430. The non-specific control sequence was 5’-GACGTAAACGGCCACAA GT-3’. A 602 subregion of BRCA1 promoter (-458 to +145) was PCR amplified from human genomic DNA using forward 5’-CTCGAGACCTTTCATTCCGCAACGCATG- 3’ and reverse 5’- AAGCTTTCCCGGGACTCTACTACCTTTAC-3’ primers (1) and subsequently subcloned into XhoI-BglII fragment of pGL4 basic vector (Promega, 1 Madison, WI). It was named as pGL4-promBRCA1. Quantitative real-time PCR All the quantitative real-time PCR were performed on 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA) using GoTaq® qPCR Master Mix (Promega, Madison, WI). The genomics region of FUT2 and PTPN4 were used as references to assess the copy number variation of TNRC9 genomic region. Primer sequences were as follows: TNRC9 exon3 forward 5’-CTTGGGGACGAGGAATTCGA-3’, reverse 5’- GTAATGGAAGGGAGGTCCA-3’; TNRC9 exon4 forward 5’-ATCGTCCACATGACC GATG-3’, reverse 5’-CTCTTCATTGATGGAGCTG-3’; FUT2 forward 5’- ATCTTCGTGGTCACCAGTAATGG-3’, reverse 5’-GGTTACACTGTGTGAGTAGAG C-3’; PTPN4 forward 5’-ACGCACGGTCCCATAGTATC-3’, reverse 5’- AAATAGTG GTGCCTCCTTGG-3’. When min (CtFUT2, CtPTPN4) - max (CtTNRC9exon3, CtTNRC9exon4) >1, the sample was considered to have a copy number amplification of TNRC9 genomic DNA. HPRT1 was used as the internal reference to assess the mRNA expression level of TNRC9. The reverse transcription of total RNA was performed by GoTaq® 2-Step RT- qPCR System (Promega, Madison, WI). The primer sequences used to amplify TNRC9 cDNA were 5’-TGCCTCACACATCTCCTTCAC-3’ and 5’- TTGGAGTCTTGGGCTTCTTGC-3’, and HPRT1 cDNA were 5’- TGACACTGGCAAAACAATGCA-3’ and 5’-GGTCCTTTTCACCAGCAAGCT-3’. The bisulfite-converted DNA was analyzed by quantitative PCR using primers for methylated BRCA1 promoter region: forward 5’-TCGTGGTAACGGAAAAGCGC-3’ (-38 to -19) and reverse 5’-AACGAACTCACGCCGCGCAA-3’ (+11 to +30) and unmethylated BRCA1 promoter region: forward 5’-TTGAGAGGTTGTTGTTTAGTGG- 3’ (-74 to -43) and reverse 5’-AACAAACTCACACCACACAA-3’ (+11 to +30) (2). The 3’ end of DHFR was used as a reference: forward 5’- TTGATGTTTAGGAGGAGAAAGG-3’ and reverse 5’- AACCCAACAATATCAAAAACTA-3’. The copies of methylated or unmethylated BRCA1 promoter were calculated using 2-(CtBRCA1-CtDHFR). The combination of methylated BRCA1 promoter and unmethylated BRCA1 promoter was used as the total copies of BRCA1 promoter. The percentage of methylated BRCA1 promoter was 2 calculated by dividing the total copies of BRCA1 promoter. Bisulfite sequencing of BRCA1 promoter To amplify a region of BRCA1 promoter, the bisulfite-converted DNA from TNRC9 abrogated ZR-75-1 cells and from control cells were amplified using a nested PCR (3). The first run was accomplished by the forward primer 5’- GGGGTTGGATGGGAATTGTAG-3’(-686 to -666 of BRCA1 gene) and reverse primer 5’-CTCTACTACCTTTACCCAAAACA-3’ (+130 to +108). One tenth of the first run amplification was re-amplified with forward primer 5’- GTTTATAATTGTTGATAAGTATAAG-3’(-592 to -568) and reverse primer 5’- AAAACCCCACAACCTATCCC-3’(+66 to +47). The second run PCR product was purified and cloned into a T vector-pGEMT (Promega). Twenty clones of each sample were randomly selected and sequenced. Immunohistochemistry Formalin fixed and paraffin embedded sections were cut at 5 micron thick and were deparaffinized and rehydrated through 3 concentrations of alcohol. Endogenous peroxidase activity was blocked with 3% H2O2 for 15 minutes. Antigen retrieval was carried out in 0.01 M citrate buffer, pH 6.0, at 95°C for 20 minutes, followed by 20 minutes cooling at room temperature. Nonspecific binding was blocked using PBST containing 5% BSA for 1 hr. Sections were incubated overnight with goat anti-TNRC9 (1:50) and mouse anti-BRCA1 antibodies (1:50) at 4 °C, followed by HRP-conjugated secondary antibodies (1:500) for 1 hour at room temperature. Antibody binding was revealed with DAB (DAKO, Denmark) as chromogen. In between steps sections were washed with PBST for 3 times, each for 7 min. Sections were counterstained with Mayer’s hematoxylin, dehydrated and mounted. Images were obtained using a bright field microscope (AxioVision software). High-grade and low-grade breast cancer samples were included in this study. Sections incubated with non-immune control sera acted as negative control. Immunoprecipitation and immunoblotting Cells were lysed with RIPA buffer (50mM Tris pH7.4, 100mM Nacl, 0.25% Na- deoxycholate, 1% NP40, 0.1% SDS) containing 1X protease inhibitors cocktail (Sigma- Aldrich, St. Louis, MO). Equal amounts of cell lysates were separated on SDS-PAGE 3 gel, transferred to PVDF membrane and probed with primary antibodies. Bands were visualized using Geliance 600 Imaging System (PerkinElmer, Waltham, MA), in conjugation with HRP-conjugated secondary antibodies and ImmobilonTM western chemiluminescent HRP substrate (Millipore, Billerica, MA). For the detection of interaction between TNRC9 and CREB, 107 transfected HEK293 cells were lysed in RIPA buffer. Lysates were precleared with Protein A/G Plus Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) and 2 mg IgG for 2 h at 4 °C. The precleared lysates were immunoprecipitated with anti-TNRC9 or anti-HA for over 4 h at 4 °C. The immunoprecipitated samples were washed three times with lysis buffer, resolved by SDS–PAGE and then subjected to immunoblotting analysis with anti-Myc. Mouse model Cells for injection are agitated slowly on a Vortex mixer in a 50 ml Falcon tube to ensure even suspension. Enough cells are then withdrawn to inoculate 5 mice; that is, ~0.5 ml of a 3x107 cell/mL suspension. The syringe is then gently inverted (containing the glass bead) to ensure even suspension. A 27 Gauge, ½ inch Precision Glide needle (BD, Franklin Lakes, NJ) is then inserted onto the syringe and primed. NU/NU nude mice (Charles River Laboratories, Wilmington, MA) are anesthetized by placing them in a 5% Isoflurane chamber. Once the mice are deeply under anesthesia, mice are removed individually, placed into the biological-safety cabinet and prepared for injection (positioned prone and upper right flank wiped with a 70% ethanol pad). The syringe is again gently inverted 3 times to ensure even cell suspension. Without tenting the skin, 0.1 ml of the cell suspension (containing approximately 3 million cells) is injected into the upper right flank of the mouse, subcutaneously. Each mouse is then placed into a separate chamber until conscious before being returned to its original cage. Tumors were monitored at 2-3 day intervals for a total of 28 days. Tumor size (width and length) was recorded, the skin overlaying the tumor was inspected and the mice weighed. After 28 days, tumors were surgically excised, measured with digital calipers and placed into 10% neutral buffered formalin. Tumors were then embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) or immunostained for Ki-67 (proliferative cells) a proliferative cell marker, using a polyclonal IgG primary antibody (rabbit; abcam) and 4 anti-rabbit biotinylated (IgG) secondary antibody (goat; Abcam) with Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) and methyl green counterstain (Vector Laboratories, Burlingame, CA). All mouse experiments were reviewed and approved by the Institutional Animal Care and Use Committee at Brown University. In silico assays for the expression of TNRC9 and BRCA1 In silico assays were performed on 266 early breast cancer patients. Transcriptional data were retrieved from NCBI GEO database (GSE21653) and further analyzed using BRBArrayTools developed by the Biometric Research Branch, National Cancer Institute (http://linus.nci.nih.gov/BRB-ArrayTools.html), Partek Genomics Suite (PARTEK, St Louis, MO), Stanford Cluster Program and TreeView software (4). Genes were projected using log2 intensity and gene ratios were average corrected across experimental samples and displayed according to uncentered correlation algorithm. Similar to breast