Supplementary Fig S3

1. A. MCF-7EpiR MCF-7TaxR siControl siSIRT6 siControl siSIRT6 Paclitaxel (h): Epirubicin (h) 0 24 48 0 24 48 0 48 72 0 48 72 SIRT6 SIRT6 FOXO3a FOXO3a p27Kip1 p27Kip1 H3K9ac Ac-p53 H3K56ac p53 H3K9Ac Ac-α-TUBULIN H3K56Ac β-TUBULIN Ac-α-TUBULIN β-TUBULIN B. MEFs MEFs WT Sirt6-/- WT Sirt6-/- Epirubicin (h): 0 4 8 16 24 0 4 8 16 24 0 4 8 16 24 Paclitaxel (h): 0 4 8 16 24 Ac-p53 Ac-p53 p53 p53 FOXO3a FOXO3a H3K9ac H3K9ac Bim Bim P-gp(MDR1) P-gp(MDR1) β-TUBULIN β-TUBULIN C. MEFs Cytoplasmic Nuclear WT Sirt6-/- WT Sirt6-/- 0 6 24 0 6 24 0 6 24 0 6 24 FOXO3a FOXO3a (low Exp) Lamin B β-Tubulin Supplementary Figure S3. (A) MCF-7-EpiR cells and MCF-7-TaxR cells transfected with siControl/siSIRT6 siRNAs were treated with 1 μM epirubicin and 10 nMpaclitaxel respectively for the times indicated, and harvested for Western Blot analysis. (B) WT and Sirt6-/- MEFs were treated with 0.1 μM epirubicin and 10 nMpaclitaxel respectively for the times indicated, and harvested for Western Blot analysis. * It is notable that some of the proteins in the Sirt6-/- MEFs appeared at lower levels. It is likely to be due to the high levels of apoptosis. (C) WT and Sirt6-/- MEFs cells were lysed in buffer A (10 mM HEPES pH 7.4, 10 mMKCl, 0.1 mM EDTA, 0.1 mM EGTA, 2mM DTT) and protease and phosphatase inhibitors, and incubated for 20 min on ice. NP-40 was added (final concentration 1%) and centrifuged. The supernatent containing the cytoplasmic fraction was kept and frozen at -70oC. The pellet was washed in buffer A and resuspended in buffer B (10 mM HEPES, 10 mMKCl, 0.1mM EDTA, 0.1 mM EGTA, 2 mM DTT, 400 mMNaCl, 1% NP-40), and rotated at 4oC for 15 min. Samples were centrifuged at 4oC and the supernatent containing the nuclear extract collected and frozen at -70oC. The cytoplasmic and nuclear extracts were separated by SDS–PAGE gel electrophoresis, transferred to Hybond-C membranes and immunoblotted with the indicated antibodies. SupplementaryFig S3