Effect of Growth/Viability Factor Deprivation on a Panel of FDC-P1 Transfectants Expressing or Not Expressing mcl-1
| Transfectant Clone . | % Viable Cells . | Total Cell No. (cells/mL ×105) . |
|---|---|---|
| I.Clones transfected with vector only | ||
| V1-CMVneo | 34 ± 9 | 5.8 ± 0.7 |
| V2-CMVneo | 6 ± 0.7 | 6.1 ± 0.4 |
| V4-CMVneo | 4 ± 2 | 5.6 ± 0.3 |
| V5-CMVneo* | 74 ± 2 | 8.0 ± 0.6 |
| V6-CMVneo | 2 ± 1 | 3.4 ± 0.8 |
| V9-CMVneo | 1 ± 0.5 | 4.4 ± 2.8 |
| II.Clones transfected with mcl-1 | ||
| IIA.Readily detectable mcl-1 expression† | ||
| S1-CMVmcl-1 | 50 ± 3 | 6.7 ± 1.1 |
| S3-CMVmcl-1 | 46 ± 4 | 6.8 ± 1.1 |
| S8-CMVmcl-1 | 43 ± 3 | 7.0 ± 1.4 |
| S13-CMVmcl-1 | 46 ± 3 | 6.8 ± 0.5 |
| S17-CMVmcl-1 | 44 ± 6 | 5.9 ± 0.7 |
| IIB.Faintly detectable mcl-1 expression‡,ρ | ||
| S21-CMVmcl-1 | 18 ± 4 | 6.2 ± 0.6 |
| S24-CMVmcl-1 | 26 ± 2 | 6.0 ± 0.4 |
| Transfectant Clone . | % Viable Cells . | Total Cell No. (cells/mL ×105) . |
|---|---|---|
| I.Clones transfected with vector only | ||
| V1-CMVneo | 34 ± 9 | 5.8 ± 0.7 |
| V2-CMVneo | 6 ± 0.7 | 6.1 ± 0.4 |
| V4-CMVneo | 4 ± 2 | 5.6 ± 0.3 |
| V5-CMVneo* | 74 ± 2 | 8.0 ± 0.6 |
| V6-CMVneo | 2 ± 1 | 3.4 ± 0.8 |
| V9-CMVneo | 1 ± 0.5 | 4.4 ± 2.8 |
| II.Clones transfected with mcl-1 | ||
| IIA.Readily detectable mcl-1 expression† | ||
| S1-CMVmcl-1 | 50 ± 3 | 6.7 ± 1.1 |
| S3-CMVmcl-1 | 46 ± 4 | 6.8 ± 1.1 |
| S8-CMVmcl-1 | 43 ± 3 | 7.0 ± 1.4 |
| S13-CMVmcl-1 | 46 ± 3 | 6.8 ± 0.5 |
| S17-CMVmcl-1 | 44 ± 6 | 5.9 ± 0.7 |
| IIB.Faintly detectable mcl-1 expression‡,ρ | ||
| S21-CMVmcl-1 | 18 ± 4 | 6.2 ± 0.6 |
| S24-CMVmcl-1 | 26 ± 2 | 6.0 ± 0.4 |
A panel of transfectant clones was tested for expression of the introduced mcl-1 gene and then assayed for cell viability under conditions of growth factor deprivation (as in Fig 5). Mcl-1 protein levels were assayed by Western blot in two separate experiments. Cell counts and cell viability (trypan blue) were assayed on day 4 in at least three separate experiments, except for clone V3-CMVneo, in which the number of experiments was two. The values shown are the mean ± SD. In one of the three experiments, the time course was followed. The time course viability profiles for clones V2-CMVneo, S3-CMVmcl-1, and S8-CMVmcl-1 were similar to those shown in Fig 6. The time course viability profiles for clones V4-, V6-, and V9-CMVneo were essentially identical to that for V2-CMVneo. Total cell number in these four clones averaged 4.2, 5.3, 6.4, and 6.0 × 105 cells/mL on days 1, 2, 3, and 4, respectively. Total cell number for clone V5-CMVneo averaged 5.3, 6.8, 8.4, and 7.8 × 105 cells/mL on days 1, 2, 3, and 4, respectively. The time course viability profiles for S1-, S13- and S17-CMVmcl-1 were essentially identical to those for S3-CMVmcl-1 and S8-CMVmcl-1. Total cell number in these five clones averaged 5.7, 6.1, 6.0, and 6.0 on days 1, 2, 3, and 4, respectively.
Two additional independent isolates of this transfectant were obtained (V3- and V7-CMVneo) and were determined to have identical bands as V5-CMVneo on Southern blots after digestion with BamHI or EcoRI and probing with CMVneo.
mcl-1 protein levels in the range of those seen in S3- and S8-CMVmcl-1 (see Fig 1B). The levels in S1-CMVmcl-1 were ∼10% higher than those in S3-CMVmcl-1 and the levels in S17-CMVmcl-1 were ∼10% lower than those in S8-CMVmcl-1.
mcl-1 protein levels estimated to be ∼20% of those seen in S8-CMV mcl-1.
ρ In separate experiments, one additional mcl-1 transfectant was tested (S15-CMVmcl-1) that had very faintly detectable mcl-1 expression and was found to exhibit 74.2% ± 2.2% viable cells (mean ± SD of 3 experiments).