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. 2015 Aug 3;6:7956. doi: 10.1038/ncomms8956

Figure 4. Birinapant co-treatment sensitized CA125-negative primary HGSC cells to carboplatin.

Figure 4

(a) DNA damage, detected by γH2Ax immunostain, was examined in CA125-negative and -positive populations from three independent primary chemo-naive human HGSCs at 12, 24 and 36 h post-carboplatin treatment. The γH2Ax foci appeared to resolve more quickly in CA125 negative compared with CA125-positive tumour cells (P=0.02 at 24 h and P<0.0001 at 36 h, unpaired two-sided t-test). Results are mean±s.e.m., n=3 HGSCs 5 fields of view each. Scale bars equal 100 μm. (b) Birinapant and carboplatin co-administration resulted in eradication of all tumour cells only in clinical samples with high cIAP levels in the CA125-negative population (results are mean±s.e.m., n=6 patients 1–4, 7 and 9 versus n=3 patients 5–6 and 8, sample run in triplicate). Cell survival was FACS quantitated based on propidium iodide and AnnexinV negativity. Western blot of pan-cIAP levels in CA125-negative and -positive subpopulations for patients 7–9 is shown. (c) Following in vitro treatment with birinapant and carboplatin co-therapy or monotherapy with either agent, primary chemo-naive human HGSC cells were re-plated in fresh media. No regrowth was observed from birinapant-sensitive HGSC (blue) following co-therapy. In contrast, growth was noted in all specimens after treatment with birinapant or carboplatin as a single agent, or after co-therapy in specimens with low levels of cIAP in their CA125-negative population (pink). Results are mean±s.e.m. (n=6 birinapant sensitive and n=3 birinapant less sensitive, samples run in triplicate). (d) CA125-negative, CA125-positive and bulk populations isolated from birinapant-sensitive primary chemo-naive human HGSC (n=3) were treated with carboplatin, birinapant or both drugs. Compared with vehicle-treated cells, carboplatin alone effectively eliminated the differentiated cells but did not kill the CA125-negative cells. On average birinapant as a single agent was equally effective against both the CA125-negative and -positive cells (P=0.085 unpaired two-sided t-test) but did not cause complete cell death in either population. Results are mean±s.e.m., n=3 HGSC run in triplicate.