American Society of Hematology

Fig. 5.

$Fig. 5. Cell death mediation by caspase-8a and -8L and inhibitory role of caspase-8L in MCF-7 cell lines. / (A) MCF-7 cells were transiently transfected with 1.5 μg β-galactosidase expression vector (pcDNA3.1/His/LacZ, Invitrogen) plus 1.0 μg empty vector (pcDNA3.1/HisA, Invitrogen), caspase-8a, and caspase-8L expression vector as described in “Material and methods.” Twenty-three hours after transfection, the extent of cell death was quantified by determining the portion of β-galactosidase–expressing cells exhibiting apoptotic morphology. They are expressed as the mean percentage of the blue cells exhibiting signs of apoptosis as a fraction of the total number of blue cells counted (about 200 cells per sample). (B) MCF-7 cells were transiently transfected with empty vector, caspase-8a, and caspase-8L expression vector using LipofectAMINE (Life Technologies) as described in “Materials and methods.” Twenty-three hours after transfection, cell lysates were fractionated by SDS-PAGE and blotted with anti–human caspase-8 antibody (MBL), which could detect intermediate form (p43 and p42) and active form (p18) caspase-8. NS indicates nonspecific bands. (C) A total of 0.5 μg caspase-8a expression vector plus various amounts of empty vector or caspase-8L expression vector were transiently transfected with 0.75 μg β-galactosidase expression vector into MCF-7 cells. The ratios of vector or caspase-8L to caspase-8a are indicated on the bottom of the graph. Twenty-three hours after transfection, apoptotic cells were determined as described in “Materials and methods.” In each experiment, the data are from 3 independent experiments. The bar indicates the SE.$

Cell death mediation by caspase-8a and -8L and inhibitory role of caspase-8L in MCF-7 cell lines.

(A) MCF-7 cells were transiently transfected with 1.5 μg β-galactosidase expression vector (pcDNA3.1/His/LacZ, Invitrogen) plus 1.0 μg empty vector (pcDNA3.1/HisA, Invitrogen), caspase-8a, and caspase-8L expression vector as described in “Material and methods.” Twenty-three hours after transfection, the extent of cell death was quantified by determining the portion of β-galactosidase–expressing cells exhibiting apoptotic morphology. They are expressed as the mean percentage of the blue cells exhibiting signs of apoptosis as a fraction of the total number of blue cells counted (about 200 cells per sample). (B) MCF-7 cells were transiently transfected with empty vector, caspase-8a, and caspase-8L expression vector using LipofectAMINE (Life Technologies) as described in “Materials and methods.” Twenty-three hours after transfection, cell lysates were fractionated by SDS-PAGE and blotted with anti–human caspase-8 antibody (MBL), which could detect intermediate form (p43 and p42) and active form (p18) caspase-8. NS indicates nonspecific bands. (C) A total of 0.5 μg caspase-8a expression vector plus various amounts of empty vector or caspase-8L expression vector were transiently transfected with 0.75 μg β-galactosidase expression vector into MCF-7 cells. The ratios of vector or caspase-8L to caspase-8a are indicated on the bottom of the graph. Twenty-three hours after transfection, apoptotic cells were determined as described in “Materials and methods.” In each experiment, the data are from 3 independent experiments. The bar indicates the SE.

This Feature Is Available To Subscribers Only