![Fig. 2. Effect of etoposide on the viability of FDC-P1 transfectants expressing or not expressing mcl-1. (A) Effect of continuous exposure to etoposide for increasing times. The 7.5-MAMmcl-1 transfectant (circles) and the 8.5-MAMneo vector-only clone (squares) were either exposed (solid symbols) or not exposed (open symbols) to dexamethasone for 2 hours at an inoculum density of 6 × 105 cells/mL. Etoposide was then applied at 20 μg/mL (time 0). Cell viability was assayed at 1, 2, and 3 days using the ethidium bromide/acridine orange method. This experiment was also performed with clones expressing mcl-1 constitutively. When clones S3- and S8-CMVmcl-1 were exposed to etoposide for 1 day, cell viability was 73% ± 14% (SD; n = 3 independent experiments) and 62% ± 13%, respectively, compared with a value of 37% ± 12% in vector-only clone V5-CMVneo. Upon exposure for 2 days, these values were 43% ± 15%, 33% ± 12%, and 17% ± 6% for S3-CMVmcl-1, S8-CMVmcl-1, and V5-CMVneo, respectively, and with exposure for 3 days the values were 29% ± 6%, 31% ± 10%, and 12% ± 3%. (B) Effect of various concentrations of etoposide. Cells were treated as in (A), except that various concentrations of etoposide were applied for 1 day. Of the nonviable cells in cultures treated for with 20 μg/mL etoposide, the fraction of cells in phases I or II of apoptosis (as a percentage of the total dead cells) was 72% ± 9.8% and 73% ± 9.7% in clone 7.5-MAMmcl-1 in the absence and presence of dexamethasone, respectively (n = 6, 3 values from the experiments shown in [A] and 3 values from the experiments shown in [B]). These values were 62% ± 5.7% and 63% ± 9.3% for clone 8.5-MAMneo in the absence and presence of dexamethasone, respectively. For both (A) and (B), the values shown are the mean ± SD of three independent experiments. / (C) Illustration of the effects of etoposide. The vector-only 8.5-MAMneo clone (left photograph) and the 7.5MAMmcl-1 transfectant (right photograph) were exposed to dexamethasone and etoposide as in (A). Cells were stained after 1 day with DAPI. The vector-only clone contains cells with condensing or fragmenting nuclei, whereas the mcl-1 transfectant contains cells with the open/lacy nuclear architecture and staining pattern typical of viable control cells. Photographs were taken at a magnification of 1,250×.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/89/2/10.1182_blood.v89.2.630/4/bl_0013f2c.jpeg?Expires=1724271382&Signature=EqRRDJ8qJD34swZyiTFHGkjeIV5zoShNY-qu3bUcve6o4rh579hOYjwXeylpDgiNzdfBLoUX5rDj2cXR6rVPPLb3RqkNo2NwcE0wond25DpEuwSc9vJouEGFjjmiFq4pikKdVZqJN7nmrUXNNICo6u7z0VoHWyXIBJlQIdaC1hzDSmqVFb-c9aeu1yyGZfr9PcLAdLBx5ka05aSSVmGsU1YDOkuc6JFaqTgOvqT7FYJfmznqQU8TgPX7ZgGoLxB8~Vb74ikwOI3gQCyIBJeRb0ykaRDpWn0QvnwA1nkkXz~GRekgwNimkY9bx3YGhjvk9qyQt7saKvJ8inntEyALqA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of etoposide on the viability of FDC-P1 transfectants expressing or not expressing mcl-1. (A) Effect of continuous exposure to etoposide for increasing times. The 7.5-MAMmcl-1 transfectant (circles) and the 8.5-MAMneo vector-only clone (squares) were either exposed (solid symbols) or not exposed (open symbols) to dexamethasone for 2 hours at an inoculum density of 6 × 105 cells/mL. Etoposide was then applied at 20 μg/mL (time 0). Cell viability was assayed at 1, 2, and 3 days using the ethidium bromide/acridine orange method. This experiment was also performed with clones expressing mcl-1 constitutively. When clones S3- and S8-CMVmcl-1 were exposed to etoposide for 1 day, cell viability was 73% ± 14% (SD; n = 3 independent experiments) and 62% ± 13%, respectively, compared with a value of 37% ± 12% in vector-only clone V5-CMVneo. Upon exposure for 2 days, these values were 43% ± 15%, 33% ± 12%, and 17% ± 6% for S3-CMVmcl-1, S8-CMVmcl-1, and V5-CMVneo, respectively, and with exposure for 3 days the values were 29% ± 6%, 31% ± 10%, and 12% ± 3%. (B) Effect of various concentrations of etoposide. Cells were treated as in (A), except that various concentrations of etoposide were applied for 1 day. Of the nonviable cells in cultures treated for with 20 μg/mL etoposide, the fraction of cells in phases I or II of apoptosis (as a percentage of the total dead cells) was 72% ± 9.8% and 73% ± 9.7% in clone 7.5-MAMmcl-1 in the absence and presence of dexamethasone, respectively (n = 6, 3 values from the experiments shown in [A] and 3 values from the experiments shown in [B]). These values were 62% ± 5.7% and 63% ± 9.3% for clone 8.5-MAMneo in the absence and presence of dexamethasone, respectively. For both (A) and (B), the values shown are the mean ± SD of three independent experiments.
(C) Illustration of the effects of etoposide. The vector-only 8.5-MAMneo clone (left photograph) and the 7.5MAMmcl-1 transfectant (right photograph) were exposed to dexamethasone and etoposide as in (A). Cells were stained after 1 day with DAPI. The vector-only clone contains cells with condensing or fragmenting nuclei, whereas the mcl-1 transfectant contains cells with the open/lacy nuclear architecture and staining pattern typical of viable control cells. Photographs were taken at a magnification of 1,250×.
