Fig 1.
Dynamic BH3 profiling assay: delta priming to PUMA-BH3.
The increase in cells primed to undergo mitochondrial outer membrane depolarisation (Δ priming) is measured by Cytochrome C release after 4 hour drug treatment. PUMA-BH3 was used at 3 μM. Values are corrected for Cytochrome C release with peptide only as described in the methods. (Mean+/- SD for n = 3).
Table 1.
Agents expected to antagonise or downregulate BCL-2 or MCL-1.
Fig 2.
Dynamic BH3 profiling assay: delta priming to (A) BAD-BH3 and (B) MS1-BH3 peptides. Delta priming is measured by cytochrome C release after 4 hour drug treatment and additional incubation with the indicated BH3 peptides (BAD-BH3 at 3 μM, MS1-BH3 at 3 μM, PUMA2A control at 100 μM). Values are corrected for cytochrome C release with peptide only as described in the methods. (Mean+/- SD for n = 3).
Fig 3.
Co-operative induction of apoptosis by ABT-199 with pladienolide B or torin1.
(A) MCL-1 long form (L) and short form (S) transcripts (i) and protein (ii) were quantified in untreated cells and cells treated with 10 nM pladienolide B (PLB) for 4 hours. (B) 4E-BP1 phosphorylation (i) and MCL-1 protein (ii) were quantified in untreated cells and cells treated with 1μM torin1 for 4 hours. (C, D) Cells were incubated with 10 nM ABT-199 (199, turquoise), 10 nM pladienolide B (PLB, red), 1 μM torin1 (green) or the indicated combinations (bright blue bar, height = effect with both agents in combination–sum of effects with agents individually). (C) After 4 hours cells were fixed and processed for Cytochrome C release. (D) After 4 hours DiOC6 was added for a further 75 minutes and 7-amino actinomycin D for the last 30 minutes. (Mean+/- SD for n = 3). Fold excess additivism (FEA) is shown on the figures and was calculated as a ratio of observed to expected values after corrections according to the Bliss algorithm (see methods). Asterisks indicate observed values significantly higher than expected values (P<0.05).
Fig 4.
Co-operative induction of apoptosis by ABT-199 with etoposide and AC220.
Cells were incubated with 10 nM ABT-199 (199, turquoise), 10 nM pladienolide B (PLB, red), 1 μM torin1 (green), 1 μM etoposide (orange) and 10 nM AC220 (mauve) or the indicated combinations (bright blue bar, height = effect with both agents in combination–sum of effects with agents individually). (A) After 4 hours cells were fixed and processed for Cytochrome C release. (B) After 4 hours DiOC6 was added for a further 75 minutes and 7-amino actinomycin D for the last 30 minutes. (Mean+/- SD for n = 3). Fold excess additivism (FEA) is shown on the figures and was calculated as a ratio of observed to expected values after corrections according to the Bliss algorithm (see methods). Asterisks indicate observed values significantly higher than expected values (P<0.05).
Fig 5.
Co-operative induction of apoptosis by pladienolide, torin1, etoposide or AC220 with JQ1.
(A) Time course of bcl-2 protein downregulation in response to JQ1 measured by flow cytometry. MFI = mean fluorescence intensity, corrected for isotype control. (B) Time course of delta priming to BAD-BH3 and MS1-BH3 measured by cytochrome C release after drug treatment and additional incubation with the indicated BH3 peptides. Values are corrected for Cytochrome C release with peptide only as described in the methods. (C) Cells were incubated with 250 nM JQ1 for 2 days. 10nM pladienolide B (PLB), 1 μM torin1, 10nM ABT-199 (199), 1 μM etoposide (ETO) or 10 nM AC220 were added for the final 4 hours. Cells were then fixed and processed for Cytochrome C release. Bright blue bar height = cytochrome C release with both agents in combination–sum of cytochrome C release with both agents individually). Fold excess additivism is shown on the figures and was calculated as a ratio of observed to expected values after corrections according to the Bliss algorithm (see methods). Asterisks indicate observed values significantly higher than expected values (P<0.05). (Mean+/- SD for n = 3).
Fig 6.
Co-operative induction of apoptosis using A-1210477.
A. Cells were co-incubated with 1 μM A-1210477 (477) and with 10 nM ABT-199 (199), 10nM pladienolide B (PLB), 1 μM torin1, 1 μM etoposide or 10 nM AC220 for 4 hours. Alternatively, cells were incubated with JQ1 for 48 hours and A-1210477 was added for the final four hours of the incubation. Cells were then fixed and processed for Cytochrome C release. (Mean+/- SD for n = 3). B. Cells were incubated with 1 μM A-1210477 for four hours and 10 nM ABT-199 was added either before, after or concurrently (final 2 hours). In the two-step conditions, cells were pelleted and rinsed twice in RPMI at 4°C in between agents. R10 = medium without drug.