SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Transcription

1 SUPPLEMENTAL EXPERIMENTAL PROCEDURES Luciferase Assays Cells were seeded on 24well plates and grown to 7% confluency. Cells were then transfected with ng of reporter constructs and 1 ng of the renilla luciferase internal control plasmid prlsv4 using Lipofectamine2 transfection protocol. Twentyfour hours after transfection, cells were treated with 5 µm or 2 nm for additional 12 hours. Cells were then lysed and subjected to DualLuciferase assay as per the manufacturer s recommendations (Promega). SUPPLEMENTARY FIGURE LEGENDS Fig. S1. HDACi have no effect on 3 S transactivation. 3 /, 3 WT or 3 S HCT116 cells were transiently cotransfected with the indicated firefly luciferase reporter constructs containing p21 or mdm2 promoter together with prlsv4 renilla luciferase vector. Cells were treated with, or and subjected to DualLuciferase assay. The results are represented as the mean ratio of firefly/renilla luciferase activities ± SD, n=3. Fig. S2. Cell death and 3 expression in response to HDACi. (A) HCT116 wild type (3 / ) and 3 / cells stably transfected with empty vector (Puro or Bsd), 3 WT or 3 S were treated with either 5 µm or 2 nm for the indicated periods of time. The percentage of cell viability was determined by trypan blue dye exclusion assay. (B) The same cell lines treated as above for 18 hours were subjected to immunoblot analysis. Fig. S3. Knockdown of mutant 3 reduces HDACiinduced apoptosis. (A) HT29 cells infected with either scrambled control (shscr) or 3 targeting shrna (sh3) lentivirus were treated with 5 μm or 2 nm for 36 hours and subjected to immunoblot and caspase3 activity assay. (B, C) SW48 and HT29 cells infected with shscr or sh3 lentivirus were treated with 5 μm or 2 nm for the indicated times, and cell viability was determined by trypan blue exclusion assay. (D) SW48 cells infected with shscr or sh3 lentivirus were treated with or 2 nm for 24 hours and subjected to Annexin VAPC/7AAD staining. Fig. S4. SirT1 does not affect induced 3dependent apoptosis. (A, B) H / and 3 D281G cells were transfected with empty pcdna3.1 or pcdna3.1sirt1 expression vectors and then treated with or 2 nm for 24 hours and subjected to immunoblot and caspase3 assays. (C, D) H / and 3 D281G cells were treated with, 1 μm EX527, 2 nm or the combination of 1 μm EX527 and 2 nm for 24 hours and subjected to western blot and AnnexinV APC/7AAD staining. Fig. S5. HCT116 3 / cells stably expressing TAP or TAP3 S were treated with 2 nm for the indicated periods of time. The percentage of cell viability was determined by trypan blue dye exclusion assay. Fig. S6. H / and 3 D281G cells were infected with shscr or shku7 lentivirus for 24 hours, treated with or 2 nm for 24 hours, and subjected to immunoblot analysis. Fig. S7. Acetylation of 3 is required for Bax translocation but not for 3 binding to BclXL. (A) HCT116 3 / cells stably expressing control Puro or 3 S were treated with, 5 μm or 2 nm for 18 hours and subjected to subcellular fractionation and immunoblot analysis. (B) HCT116 3 / cells stably transfected with empty (Puro), M3 S or M3 S3KR were treated with, 5 μm or 2 nm for 16 hours and subjected to subcellular fractionation and immunoblot analysis. (C, D) HCT116 3 / cells were transiently transfected with BclXL and the indicated Mtagged 3 expression plasmids or empty control vector for 36 hours, treated with or

2 without 2 nm for additional 12 hours, and subjected to immunoprecipitation in CHAPS lysis buffer with antim monoclonal antibody, followed by immunoblot analysis with the indicated antibodies.

3 Firefly/Renilla Empty p21 MDM2 Empty p21 MDM2 Empty p21 MDM2 WT) 3 / Figure S1

4 A Viability (%) Time (h) Time (h) / WT) 3/ Puro 3/ Bsd Puro Bsd 16 Bsd 63 Bsd 71 3 / B 3 / WT) Puro Bsd Puro Bsd 16 Bsd 63 Bsd 71 (clone) 3 βactin Figure S2

5 A B HT29 Empty shscr sh3 % Viability 9 βactin 3 Caspase3 Activity ( FU/hr/mg protein) % Viability.36 ±.1 FL3H::7AAD ± ±.3 13 FL4H::AnnexinVAPC 2.3 ± ± FL4H::AnnexinVAPC ± ± ±.39 sh3.41 ±.13 Hours FL3H::7AAD ± ± Empty Empty shscr shscr sh3 sh3 9 shscr 48 SW48 24 Hours HT C D FL3H::7AAD 7 8 FL3H::7AAD 8 6 Empty shscr sh3 Empty Empty shscr shscr sh3 sh3 12 SW ± FL4H::AnnexinVAPC 1.11 ± ± ± ± FL4H::AnnexinVAPC 14 Figure S3

6 3 / SirT1 D281G) B βactin SirT1 3 AcK3823 C 3 / Caspase3 Activity ( FU/mg Protein/hr) A D D281G) 3 / 3 /D281G) Empty Empty SirT1 SirT1 D281G) AcK βactin EX527 EX527 EX527 Figure S4

7 Viability (%) TAP TAP Time (h) Figure S5

8 3 / D281G) shscr shku7 Ku7 βactin Figure S6

9 WCL Figure S7 3 BclXL IP: M Ku7 BclXL M3 M M IP: M WCL S3K R) S3K R) y pt D Em M Mitochondria ) KR S W Q/ Pu S KR ro ) L 22 W Cytoplasm M y C pt Q/ Pu ro L 22 B Em Em p M ty M W M 3 T ) (L p Q (L Em 22 2 Q/ 3S pt M y W ) Sp M 5 3K R) p W M 53 T ) (L p Q (L 22 2 Q/ 3S W ) S3K R) Pu ro Pu ro A Bax 3 Ku7 Histone H3 αtubulin Nucleus Bax HSP6 αtubulin Cytoplasm