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. 2004 Jul;78(13):7097–7111. doi: 10.1128/JVI.78.13.7097-7111.2004

FIG. 5.

FIG. 5.

M11L efficiently blocks the release of cytochrome c and PARP cleavage induced by FasL in HEKM11L cells. (A) Expression of M11L was shown in stably expressing HEK293 cells (HEKM11L) by immunoprecipitation. (B) HEKneo and HEKM11L cells were either left untreated or treated with FasL (at 4 or 10 ng/ml) to determine cell death numbers. At various times posttreatment, the stable cell population was stained with trypan blue. The percentage of blue cells within the total cell population was determined from four replicates of 300 cells each. (C) M11L expressed from HEKM11L cells inhibits PARP cleavage in response to FasL. Total cell lysates (50 μg/lane) were analyzed by immunoblotting with anti-PARP, which indicated that M11L protected most of the PARP from cleavage, unlike controls exposed to FasL. (D) (Top panel) Analysis of cytochrome c release in HEKM11L and control HEKneo cells following FasL treatment. The HM fraction, enriched for mitochondria, was separated from the soluble cytosolic fraction. Cytochrome c cleavage was not detectable in the cytosolic fraction in either cell line; this is considered the uninduced basal level (lanes 1 and 2). However, upon induction with FasL, there was a significant loss of cytochrome c from the HM fractions of control cells (lane 7) relative to that in stable cells (lane 8). This result corresponds with an increased appearance of cytochrome c in the cytosolic fraction for controls (lane 3) versus cells expressing M11L (lane 4). (Bottom panels) Loading controls (actin and COX IV) for each fraction. (E) Measurement of pro-caspase 9 decrease in stable cells. (Right) Lanes 1, 3, and 5, HEKneo cells; lanes 2, 4, and 6, HEKM11L cells. Samples were either left untreated (lanes 1 and 2) or treated with FasL at 10 ng/ml (lanes 3 and 4) or 4 ng/ml (lanes 5 and 6). (Left) Differences in pro-caspase 9 band intensity were calculated after normalization against the actin loading control and plotted on a bar graph as a representation of protection by M11L from FasL-mediated apoptosis.