Figure 3.
Progressive transformation of SA′ cells in serial assays for focus formation and the sensitivity to MTX of actively multiplying transformed cells. Freshly thawed SA′ cells (105) were seeded in MCDB 402 with 10% CS, which was switched to 2% CS at 3 days for an additional 11 days before fixation and staining (1°, 105). Cells (104) from a sister culture were mixed with 105 standard passage A′ cells for a 2° assay (2°, 104). They also were seeded at 105 cells in the 2° assay, and these were subcultured after 14 days for a 3° assay at 105 alone (3°, 105) and 103 with 105A′ cells (3°, 103). Half of the cultures in the 3° assay were treated from days 3 through 6 with 200 nM MTX and then incubated 8 more days without MTX (3°, 105) and (3°, 103) before staining. Sister cultures from the untreated and MTX-treated 3° assay of 103 cells were reseeded at 105 for a 4° assay in the absence of MTX (4°, 105). Cultures that were untreated in the 3° assay are on the left in both the 3° and 4° assay, and the MTX-treated 3° assay and their 4° descendants are on the right. The arrows connect the untreated and MTX-treated 3°, 103 cultures with their respective 4°, 105 subcultures.