Fig 1.
CHX induces a time and dose-dependent Bax/Bak-dependent death in FDM cells.
A) WT and bax/bak DKO cells were treated with CHX at the concentrations indicated for 24 h before determining cell viability by PI exclusion and flow cytometry. bax/bak (B) and WT (C) cells were treated with 20 μg/ml CHX for the times indicated before cell viability was determined. D) WT cells were incubated with 20 μg/ml CHX for the times indicated before being washed and resuspended in fresh media without CHX. Cultures were continued before cell viability was determined at 24 h. Data presented are expressed as mean ± SEM from 3 independent experiments.
Fig 2.
CHX induces death in FDM cells independently of several known BH3-only proteins.
Viability of (A) WT and bax/bak DKO, (B) single BH3-only knockout and (C) BH3-only DKO FDM cell lines were determined after 24 h of treatment with 20 μg/ml CHX. (D) WT, bax/bak and BH3-only knockout cells were treated with 20 μg/ml CHX for the times indicated before cell viability was determined. Data shown is expressed as mean ± SEM from 3 independent experiments. The numbers after a cell line’s name refer to independent clonal lines of the same genotype.
Fig 3.
CHX induces the proteasomal loss of Mcl-1, but not other pro-survival Bcl-2 family proteins.
A) Lysates of WT and bax/bak DKO cells treated with 20 μg/ml CHX for 0–8 h were separated by SDS-PAGE, transferred to membranes and immunoblotted with antibodies to Mcl-1, Bcl-2, Bcl-xL and Bcl-w. 293T cells transiently expressing mouse Bcl-xL, and FDCP1 cells expressing Bcl-w, were used as positive controls for the Bcl-xL and Bcl-w antibodies. ß-actin was used a loading control. B) Densitometry analysis of western blots probed with pro-survival proteins, normalized to 0h timepoint. Mcl-1 densitometry refers only to the transient form of the protein, in bax/bak DKO cells. Results are representative of three independent experiments. C) WT and bax/bak DKO cells were pre-incubated with or without 20 μM MG132 before being treated with or without 20 μg/ml CHX as indicated. Cell lysates were immunoblotted for Mcl-1 and ß-actin as shown. Asterisks indicate cleavage products of Mcl-1 and cross-reactive species.
Fig 4.
Roscovitine reduces levels of Mcl-1 and induces death in FDM cells independently of BH3-only proteins.
A) Lysates of WT and baax/bak DKO FDM cells treated with roscovitine (30 μM) for the indicated times were separated by SDS-PAGE, transferred to membrane and immunoblotted with the Mcl-1. β-actin was used a loading control. B) WT cells were treated with increasing concentrations of roscovitine before cell viability was determined. C) WT, bax/bak and BH3-only knockout cells were treated with 30 μg/ml roscovitine for 24 h before cell viability was determined. Data shown is expressed as mean ± SEM from 3 independent experiments. The numbers after a cell line’s name refer to independent clonal lines of the same genotype.
Fig 5.
IL-3 withdrawal reduces Mcl-1 abundance.
A) WT and bax/bak DKO cells were washed and incubated in media without IL-3. Cell viability was determined at 24 and 48 h after IL-3 removal. Data shown are from a single experiment. B) Cell lysates were prepared at 0, 24 and 48 h following IL-3 withdrawal and immunoblotted for Mcl-1 and ß-actin as shown.
Fig 6.
Mcl-1 over-expression gives partial protection to wild-type cells from CHX-induced cell death.
A) Clones of WT FDM cells bearing inducible Mcl-1 or Bcl-2 expression constructs were treated for 24 h with or without 100 nM 4HT to induce Mcl-1 or Bcl-2. Cell lysates were prepared and immunoblotted with anti-Mcl-1, anti-flag (to detect flag-Bcl-2) or ß-actin as indicated. B) The same cells as in (A) were treated with 100 nM 4HT for 24 h before being culture in the presence or absence of 20 μg/ml CHX for a further 24 h before cell viabilities were determined by PI exclusion and flow cytometry. Data shown is expressed as mean ± SEM from 3 independent experiments. The numbers after a cell line’s name refer to independent clonal lines of the same genotype.
Fig 7.
Bak deficient FDM cells are resistant to CHX-induced apoptosis.
Cell viabilities of WT, bax/bak DKO, and bax and bak SKO FDM cell lines were determined after 24 h of treatment with 20 μg/ml CHX. Data shown is expressed as mean ± SEM from 3 independent experiments. The numbers after a cell line’s name refer to independent clonal lines of the same genotype.
Fig 8.
BimS2A induces efficient cell death in wild-type FDM cells.
A) Inducible bax/bak DKO FDM cells were treated for 24 h with or without 100 nM 4HT to induce BimS2A expression as indicated. Cell lysates were prepared and immunoblotted with anti-flag to detect flag-BimS2A or ß-actin as indicated. B) Inducible WT and bax/bak DKO FDM cells were treated with or without 100 nM 4HT to induce BimS2A expression as indicated. Cell viabilities were determined 24 h after the addition of 4HT. Data shown is expressed as mean ± SEM from 3 independent experiments.