Suplementary Methods
Lentiviral shRNA. Stable expression vectors for small hairpin RNA (shRNA)
were constructed by ligating annealed oligonucleotides (GATCCG sense siRNA
TTCAAGAGA antisense siRNA CTTTTTTGGAAA) containing siRNA sequences
to BIMEL (CCGAGAAGGTAGACAATTG) (Reginato et al., 2003), or a mismatch
control (BIM-MM, CCGAGTAGCAAGAGAATTG) into the pLVTH lentiviral vector
(Wiznerowicz & Trono, 2003). HEK293 cells were transfected with 6µg pLVTHM
containing shRNA sequences, 3µg pMD.VSVG, 3µg pCMVdR8.91 using
Lipofectamine2000 (Invitrogen). Following overnight incubation at 37ºC in 5%
CO2, the medium was replaced with fresh DMEM. The virus-containing medium
was harvested 48 hours after transfection, centrifuged (3000rpm, 15 mins) and
passed through a 0.45µm filter. Live virus was added to HT29 cells at 40%
confluency in 24-well dishes. Clones were obtained by serial dilution and shRNA
expressing clones were identified by analysing EGFP expression by flow
cytometry and western blotting for BIM.
Legends to Supplementary Figures.
Figure S1. Expression of endogenous
V600EBRAF
suppresses apoptosis in
response to serum withdrawal. (A) BrafLSL-V600E and Braf+/+ MEFs containing
the CreERTM allele were treated with or without 4-HT for 96 hours after which
they were treated for 48 hours in the presence (FBS) or absence (SF) of growth
factors. Cells were then fixed and stained with Hoechst 33258 and analysed by
fluorescence microscopy. Treatment of the Braf LSL-V600E;CreERTM cells with 4-HT
induces BRAFV600E expression and this leads to a significant reduction in the
proportion of cells displaying nuclear shrinkage. (B) BrafLSL-V600E MEFs containing
the CreERTM allele were treated with or without 4-HT for 96 hours after which
they were treated for 24 hours in the presence (FBS) or absence (SF) of growth
factors. Cells were subjected to a DEVDase assay as described in Materials and
Methods. Data presented is the mean ± SEM of three experiments. Induction of
BRAFV600E expression led to a statistically significant reduction in caspase activity
following serum withdrawal.
Figure S2. Caspase-independent cell death in COLO205 and LS411 cells.
(A) Colo205, HT29, LS411 and CO115 cells were maintained in FBS (white bars)
or serum starved in the presence of U0126 (black bars) with or without
zVAD.fmk. Caspase inhibition protected HT29 and CO115 cells but not Colo205
and LS411 cells. (B) The inability of zVAD.fmk to protect Colo205 cells was not
due to defective zVAD.fmk since it completely prevented activation of
DEVDase/caspases in these cells. (C) Differences in sensitivity to zVAD.fmk
between HT29 and Colo205 cells are not due to differences in inactivation of
ERK1/2 or expression of BIM following MEK inhibition.
Figure S3. De-phosphorylation and expression of BIM in CO115 and LS411
cells. (A & B) LS411 and CO115 cells were serum starved alone (SF) or with
U0126 (SF+U0); some cells were also treated with U0126 in the presence of FBS
(FBS+U0). Whole cells extracts were fractionated by SDS-PAGE and probed by
Western blot. Serum withdrawal fails to cause de-phosphorylation or increased
expression of BIM but this is overcome by U0126. Indeed, MEK inhibition is
sufficient to induce BIM even in cells maintained in FBS. (C) HT29, COLO205
and CO115 cells were left untreated or treated with U0126. Western blotting of
whole cell extracts revealed that all three cell lines have high P-ERK1/2 but only
CO115 cells have high P-PKB. Induction of BIM in CO115 cells in (B) occurs
regardless of this high PKB signalling in CO115 cells (C), which might normally
repress BIM transcription via FOXO3A phosphorylation.
Figure S4. Cycloheximide protects HT29 cells against death arising from
MEK inhibition. (A) HT29 cells were pre-treated with cycloheximide as indicated
and then subject to serum starvation in the presence of U0126. Activation of
caspase following MEK inhibition was abolished by cycloheximide indicating that
turnover of a labile protein contributes to death. (B) Whole cells extracts
prepared in parallel to (A) were fractionated by SDS-PAGE and probed by
Western blot. Cycloheximide reduced basal levels of MCL-1 and BIM. It also
reduced the expression of BIM induced by MEK inhibition. The reduction of BIM
correlates with the protective effects of cycloheximide whereas the loss of MCL-1
would be expected exacerbate cell death - this was not seen.
Figure S5. Stable shRNA-mediated silencing of BIM protects HT29 cells
against death induced by MEK inhibition. Wild-type HT29 cells and HT29
cells expressing shRNA to BIM (BIM) or a mismatch control (BIM-MM) were
treated with medium containing growth factors (F, FBS), deprived of growth
factors in the presence of 20µM U0126 (SF+U0) or 1µg/ml Cisplatin (C,Cis). The
expression of BCL-2 family members was determined by Western blotting (A)
and the percentage of cells with sub-G1 DNA was determined by PI staining and
flow cytometry (B). Results are combined from at least three experiments
performed in duplicate and are the mean ± SEM. * p<0.05 relative to WT; **
p<0.01 relative to FBS control.
(C) HT29 cells were treated with media
containing serum (FBS) or serum starved (SF) in the absence or presence of
20µM U0126 for 4 hours.
RNA was isolated and real time qRT-PCR was
performed as described in the materials and methods. BIM mRNA levels were
normalised to that of GAPDH and the result is the mean ± SEM of four
experiments