Cells and Spleen Cell Transformation Assays

Supplemental Material

Cells and spleen cell transformation assays

Transformed chicken spleen cells, BJAB EBV (-) Burkitt type lymphoma cells, Daudi EBV (+) Burkitt type lymphoma cells, RC-K8 diffuse large B-cell lymphoma cells, SUDHL4 diffuse large B-cell lymphoma cells, IB4 EBV (+) umbilical-cord B-cell lymphoblastoid cells, BL41 Burkitt lymphoma cells, MedB-1 mediastinal B-cell lymphoma cells, Karpas1106 mediastinal B-cell lymphoma cells (a gift of M Dyer, Leicester University, UK), L428 Hodgkin’s lymphoma cells, HDLM2 Hodgkin’s lymphoma cells, HeLa human cervical cancer cells, HCT15 human colon carcinoma cells, SW260 human colon carcinoma cells, MCF7 human breast cancer cells, Colo205 human colorectal cancer cells, HT29 human colon adenocarcinoma cells, A293 human embryonic kidney cells, chicken DF1 cells and mouse 3T3 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10-20% fetal bovine serum, 50 units/ml penicillin, and 50 mg/ml streptomycin.

Conditions and primers for RT-PCR

For PCR on 2 ml of cDNA, the conditions were as follows. GAPDH PCR of cell line cDNA consisted of 18 cycles of 94 °C denaturation (30 s), 60 °C annealing (30 s) and extension (30 s); GAPDH PCR of tissue sample cDNA consisted of 25 cycles of the same program. For both cell line and primary tissue cDNA, PCR of REL and RELD9 consisted of 2 cycles of 94 °C denaturation (1 min), 65 °C annealing (45 s) and extension (1 min), 2 cycles of 94 °C denaturation (1 min), 64 °C annealing (45 s) and extension (1 min), 2 cycles of 94 °C denaturation (1 min), 63 °C annealing (45 s) and extension (1 min), 27 cycles (REL) or 36 cycles (RELD9) of 94 °C denaturation (1 min), 62 °C annealing (45 s) and extension (1 min). PCR of REL, REL+Alu and RELD9 together (Figure 5b) was done using the above program for 36 cycles.

Splice variant-specific primers used for cDNA amplification of REL were 5’-GCTATCACAGAACCCGTAACAG-3’ (sense) and 5-CCATGACTGTTTGGATTAGTACTGTTTG-3’ (antisense), resulting in a 919-bp product, and of RELD9 were 5’-AAACTGTGCCAGGATCACGAACC-3’ (sense) and 5’-CCATGACTGTTTGGATTAGTACTGTTTG-3’ (antisense), resulting in a 702-bp product. For co-amplification of REL, REL+Alu, and RELD9, the primers 5-GCTATCACAGAACCCGTAACAG-3’ (sense) and 5’-ACCCCTGTAGGCATTTCTCTCACA-3’ (antisense) were used, resulting in three products of 290, 386, and 221-bp, respectively. For GAPDH, 5-TGGTATCGTGGAAGGACTCATGAC-3’ (sense) and 5-ATGCCAGTGAGCTTCCCGTTCAGC-3’ (antisense) were used, resulting in a 189-bp product.