TABLE 3.
Effect of antisense IRF-4 on proliferation and soft agar colony formation of v-Rel-transformed cellsa
| Virus | Cells | Mean proliferation (106 cells)b ± SD | Mean saturation density (106 cells/cm2)c ± SD | Mean no. of colonies in soft agard ± SD |
|---|---|---|---|---|
| None | CEF | 2.53 ± 0.15 | 0.35 ± 0.03 | 0 |
| DS3 + CSV | CEF | 2.56 ± 0.25 | 0.37 ± 0.03 | 0 |
| DS3 + REV-anti-IRF-4 | CEF | 0.63 ± 0.12 | 0.16 ± 0.02 | 0 |
| DSv-Rel + CSV | CEF | 2.40 ± 0.18 | 0.32 ± 0.03 | 65.4 ± 12.2 |
| DSv-Rel + REV-anti-IRF-4 | CEF | 0.83 ± 0.15 | 0.14 ± 0.04 | 0 |
| CSV | C4-1 | 90.3 ± 12.7 | NT | NT |
| REV-anti-IRF-4 | C4-1 | 15.6 ± 5.5 | NT | NT |
CEF cultures were infected with empty retroviral vector DS3 or a retrovirus expressing v-Rel (DSv-Rel) at a multiplicity of infection of 3. Control CEF cultures were left uninfected. Four weeks after infections, DS3- or DSv-Rel-infected fibroblasts were superinfected with REV–anti-IRF-4 or CSV 6 h after plating cells for proliferation and saturation density assays (see below). Since ALV-derived DS3 and DSv-Rel use different receptors to gain entry to the cell than reticuloendotheliosis virus-derived REV–anti-IRF-4 with CSV helper virus, the viruses did not interfere, and superinfection was highly efficient. For soft agar colony formation assays, DS3- or DSv-Rel-infected fibroblasts were superinfected 24 h before plating in soft agar medium. The lymphoid cell line C4-1, transformed by S2A3 v-rel, was infected with REV–anti-IRF-4 or CSV.
Fibroblasts from each culture were plated (2 × 105 cells per 60-mm-diameter dish) or C4-1 cells were seeded (2 × 106 cells in 5 ml of culture medium per bottle), and the number of cells was determined after 48 h by counting with a hemacytometer. The mean ± standard deviation for three independent experiments is shown.
Fibroblasts from each culture were plated on six 60-mm-diameter dishes (2 × 105 cells per dish), and the medium was changed each day. The number of cells was determined each day by counting with a hemacytometer until maximum density was reached. The mean ± standard deviation for three independent experiments is shown.
CEF cultures (105 cells) were seeded in soft agar (0.37%) on top of a bed of hard agar (0.75%). The plates were scored for the development of colonies 3 weeks after seeding. The mean ± standard deviation for three independent experiments is shown. NT, not tested.