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. Author manuscript; available in PMC: 2018 Jul 10.
Published in final edited form as: Nature. 2018 Jan 10;553(7688):351–355. doi: 10.1038/nature25170

Extended Data Figure 3. Attenuation of oxidative stress does not affect REV-ERBs agonists’ cytotoxic activity.

Extended Data Figure 3

a–b, REV-ERBs agonists treatment induce apoptosis also upon co-treatment with NAC and under hypoxic condition as showed by proliferation assay of HCT-116; n= biological replicates n=3 (mock ± NAC), n=6 (09–011 ± NAC), n=9 (09 + NAC), n=11 (011 + NAC) 20 µM, n=3 (mock normoxia), n=6 (09–011 normoxia/hypoxia), n=5 (mock hypoxia) 6 days, one-way ANOVA test ****P<0.0001). c–d HCT116 apoptosis induction remained unchanged under hypoxia or upon co-treatment with NAC; 20 µM 6 days, one-way ANOVA test, n=biological independent samples n=5 (mock), n=6 (SR9009), n=8 (SR9011) normoxia TUNEL ***P=0.0003; normoxia Cl. Caspase 3 **P=0.0021; n=3 (mock), n=5 (SR9009), n=4 (SR9011) hypoxia TUNEL, **P=0.0015; hypoxia Cl. Caspase 3 **P=0.0046; NAC TUNEL, ****P<0.0001; NAC Cl. Caspase 3 ****P<0.0001) e, also in MCF-7 apoptosis triggered by REV-ERBs agonists is oxidative state independent, as showed by proliferation assay (20 µM n= biological replicates n=6 (mock normoxia/hypoxia), n=4 (09–011 normoxia), n=7 (09 hypoxia), n=5 (011 hypoxia) one-way ANOVA test ****P<0.0001; f–g TUNEL assay and immunofluorescence analysis of Cleaved Caspase 3 confirm previous results; n= biological independent samples n=3 (mock normoxia), n=5 (mock hypoxia), n=11 (09 normoxia), n=8 (09 hypoxia), n=10 (011 normoxia/hypoxia), One-way ANOVA test TUNEL normoxia **P=0.0049; Cl. Casp. 3 normoxia **P=0.0054; TUNEL hypoxia ****P<0.0001; Cl. Casp. 3 hypoxia ****P<0.0001; all panels three biological independent experiments with similar results. All the data are plotted as mean ± s.e.m. Norm= Normoxia; Hypo= Hypoxia.