Northern blot analysis of mRNA extracted from BC-3 cells uninduced or TPA induced for 48 h in the presence of MTX. (A) RNA was hybridized with viral DHFR, cellular DHFR, c-fos, and human β-actin probes. Lane 1, uninduced; lane 2, TPA induced; lane 3, uninduced and treated with 1 μM MTX; lane 4, TPA induced in the presence of 1 μM MTX; lane 5, TPA induced and 24 h after induction treated with 1 μM MTX for an additional 24 h; lane 6, TPA induced BJAB. (B) mRNA was hybridized with ORF50/Rta, kaposin/K12, viral IL-6, viral GPCR, viral MIP II, MCP, viral FLIP/cyclin, and human β-actin probes. Lane 1, uninduced; lane 2, TPA induced; lane 3, uninduced and treated with 1 μM MTX for 48 h; lane 4, TPA induced for 48 h in the presence of 1 μM MTX; lane 5, TPA induced and 24 h after induction treated with 1 μM MTX for an additional 24 h; lane 6, TPA induced BJAB. (C) Kinetics of ORF50/Rta. BC-3 cells were induced with TPA for 48 h, and 1 μM MTX was added 0, 4, 8, 12, and 24 h after induction. Lane 1, uninduced; lane 2, TPA induced; lane 3, TPA induced and MTX added 0 h after induction; lane 4, TPA induced and MTX added 4 h after induction; lane 5, TPA induced and MTX added 8 h after induction; lane 6, TPA induced and MTX added 12 h after induction; lane 7, TPA induced and MTX added 24 h after induction. (D) Northern blot analysis of mRNA extracted from BC-3 and MTX-R-BC-3 cells. RNA was hybridized with ORF50/Rta, viral DHFR, viral GPCR, K12/kaposin, viral IL-6, human β-actin, c-fos, and cellular DHFR probes. Lane 1, BC-3; lane 2, TPA induced BC-3; lane 3, 0.5 μM MTX-R-BC-3 cells maintained in 0.5 μM MTX; lane 4, 1 μM MTX-R-BC-3 cells maintained in 1 μM MTX; lane 5, 0.5 μM MTX-R-BC-3 cells, TPA induced in the presence 0.5 μM MTX; lane 6, 1 μM MTX-R-BC-3 cells, TPA induced in the presence 1 μM MTX; lane 7, 1 μM MTX-R-BC-3 cells, TPA induced in the absence of 1 μM MTX; lane 8, TPA induced BJAB.