Figure S1 (related to Fig. 1): The prototypical mitochondrial pathway of apoptosis is involved in cell-death of v-src-transformed cells.
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1 Figure S1 (related to Fig. 1): The prototypical mitochondrial pathway of apoptosis is involved in cell-death of v-src-transformed cells. (A) Non-transformed (Control cells) and v-srctransformed 3T3 cells expressing active Src (v-src 37 C) or heat-inactivated Src (v-src 39.5 C) were exposed to a variety of apoptosis-inducing stimuli and caspase-3 activation was detected by anti-cleaved caspase-3 Western-blotting on whole cell lysates. (B) Mitochondria isolated from non-transformed (Control) and v-src-transformed 3T3 cells with either active (v-src 37 C), or heat-inactivated Src (v-src 39.5 C) were incubated with 10 nm tbid for the indicated times and recovered by centrifugation. Cyt-c release was revealed by anti-cyt-c Western blot of mitochondria pellets and supernatants. These results indicated that v-src-transformed 3T3 cell resistance to apoptosis did depend on Src activity. (C) Bax mitochondrial relocation and activation upon staurosporine treatment were revealed by immunostaining of activated Bax with mab5b7 (red) in control cells but not in v-src cells. (D) tbid-induced Bax insertion into mitochondria isolated from control cells was assessed by anti-bax Western blots after alkali treatment (Na2CO3) of pellets previously incubated with 10 nm tbid for the indicated times. First lane shows the total amount of Bax present in isolated mitochondria. Under these conditions, no Bax insertion is observed in mitochondria from v-src cells. (E) Isolated iitochondria from control and v-src-transformed cells were incubated with increasing concentrations of tbid for 30 min. Then, anti-tbid Western blots were performed on pellets and supernatants to reveal the amount of inserted tbid. These results showed that tbid bound to mitochondria from v-src-transformed cells and control cells in an identical manner. (F) Isolated mitochondria from v-src-transformed cells were incubated with increasing concentrations of tbid for 30 min, as in Fig. 1B. Then, mitochondrial pellets were treated with sodium carbonate (Na 2 CO 3 ) and Bax insertion was checked by anti-bax Western blots of the resulting pellets. These results showed that the cyt-c release machinery was functional in mitochondria of v-src-transformed cells, that it depended upon Bax activation, but that higher concentrations of tbid were required to activate it.
2 Figure S2 (related to Fig. 2): (A) Contributions of Bik and Bad to the release of cyt- c from isolated mitochondria from 3T3 untransformed cells (Ctrl Mitochondria) were studied by incubating mitochondria with increasing tbid concentrations with vehicle (Mock) or anti- Bik BH3- domain antibodies or anti- Bad Y208 antibodies. The tbid concentration allowing cyt- c release (*) decreased significantly when Bik, but not Bad, was inhibited. (B) Control cells were transfected with shrna against Bik, Bad, Puma or Bim. Knockdown of endogenous proteins were validated by western-blotting. (C) Control and v-src-transformed cells transiently transfected with a HA-Bik expression plasmid for 36 hours were treated for 6 hours with either vehicle or staurosporine (Stauro). HA-Bik (red) was revealed by immunostaining with anti-ha antibody and nuclei were stained by Hoechst Percentages of cells expressing HA- Bik with pyknotic nuclei are indicated (mean ± SD (in brackets) in three independent experiments). These results show that v-srctransformed cells that express Bik undergo normal apoptosis upon staurosporine induction.
3 Figure S3 (related to Fig. 4): (A) Validation of inhibitors used in Fig.4A. v-src-transformed cells were treated as described in Fig 4A. Lysates were analyzed by western-blotting for phospho- and total amount of substrates of the targeted kinases. (B) Control of expression of plasmids used in Fig. 4B and 4D. (C) Validation of inhibitors and activators of the RAS-RAF-MEK1/2-ERK1/2 pathway used in Figs. 4A and 4B.
4
5 Figure S4 (related to Fig. 6): (A) Bik mrna expression was evaluated by RT-PCR in the four human cell lines. GAPDH expression was used as a control. LS174T do not express BIK. (B) COLO205 were treated overnight with BRAF inhibitor PLX4720. Endogenous BIK and activation of ERK1/2 (perk1/2) were monitored by western-blotting. Inhibition of BRAF restores BIK expression in these cells. (C) Validation of BIM and BIK knockdown in the four human cell lines. Cells were previously treated with PD in order to re-express BIK. (D) Apoptosis was triggered by thapsigargin in NCI-H460, COLO205, HCT116 and LS174T cells silenced for bik or/and bim. SRC was inhibited by dasatinib when indicated. Apoptosis was restored by dasatinib in NCI-H460 and LS174T cells and depended respectively on BIK and BIM. Dasatinib failed to resensitize COLO205 and HCT116. (E) Apoptosis was triggered as in (D) but upon PD treatment instead of SRC inhibition. MEK1/2 inhibition restored sensitivity to apoptosis in NCI- H460 where it depended on BIK and in COLO205 and HCT116 where it depended on both BIK and BIM. In COLO205, BRAF inhibition by PLX4720 was similarly able to resensitize cells to apoptosis.
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