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Suplementary experimental procedures:

Suplementary experimental procedures: Measurement of reactive oxygen species (ROS): ROS were measured as described by Regazzetti et al. {Regazzetti, 2009 #672} using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Molecular Probes).

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Suplementary experimental procedures:

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  1. Suplementary experimental procedures: Measurement of reactive oxygen species (ROS): ROS were measured as described by Regazzetti et al. {Regazzetti, 2009 #672} using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Molecular Probes). Viability assay: Cells were seeded at 2.103 per well in 96-well plates and incubated in medium containing 10% FBS.24 hours after seeding, cells were treated 2-DG (20mM) or cultivated in HBSS. After 3 days, XTT was added to the wells, cells were incubated for 4h at 37°C and the optical density was measured.

  2. B A PC3 DU145 A549 0 2-DG 0 2-DG PARP c PARP c Caspase 3 activity (relative activity/min./mg prot.) 0.7 Erk2 Erk2 0.6 0.5 0.4 0.3 0.2 Fig. S1: (A) DU145 and PC3 cells were treated with 20mM 2-DG for 48 hours and PARP was analysed by western blotting. (B) Measurement of caspase 3 activity in A549 (non small cell carcinoma cell line) treated with 20mM 2-DG or 1µM staurosporin. 0.1 0 0 0 2-DG 2-DG STAU

  3. 2-DG siAkt1/2 0 siAkt1/2 0 siCt 2-DG siCt 56,80% 34,80% 13,96% 39,38% 49,74% 67,17% 10,64% 19,21% Propidium iodide Annexin V Fig. S2: Flow cytometry analysis of LNCaP cells transfected with siCt or siAkt1/2, treated with 2-DG 20mM for 24h, and labelled with Annexin V. The percentage of cells positively labelled with Annexin V is indicated for each condition.

  4. A siCt siSesn2 2-DG 0 2-DG 0 Sesn2 MEF Sesn2 +/+ MEF Sesn2 -/- C M5 M10 Rapa C M5 M10 Rapa P-4EBP1 Sesn2 P-S6 ribo 4EBP1 P-4EBP1 tubulin 4EBP1 B tubulin Fig. S3: (A) LNCaP cells transfected with siRNA Ct or siRNA Sesn2 were treated or not with 20mM 2-DG for 24 hours and an immunoblot of the indicated protein was performed. (B) WT or Sesn2 invalidated MEFs were treated with Rapamycin (40nM) or 5mM (M5) or 10mM (M10) metformin for 24 hours and an immunoblot of the indicated protein was performed.

  5. HBSS 2-DG 20 mM 4.5.10-6 3.5.10-6 2.5.10-6 A UA fluo.moL-1 B MEF P53 +/+ MEF P53 -/- C 2-DG C 2-DG 1.5.10-6 p53 Sesn2 5.10-5 0 Tubulin +Nac 8h 0 Nac 1mM 8h 8h H2O2 500µM Fig. S4: (A) WT or p53 invalidated MEFs were treated or not with 20mM 2-DG for 24 hours and an immunoblot of the indicated protein was performed. (B) Measurement of reactive oxygen species (ROS) in LNCaP cells treated with 500µM H2O2 (inducer of ROS) for 8 hours, N-acetylcystein (Nac) (inhibitor of ROS), 20mM 2-DG and incubated with HBSS for the indicated time.

  6. LNCaP PC3 DU145 C 2-DG Akti 2-DG+Akti C 2-DG Akti 2-DG+Akti Sesn2 Sesn2 P-Akt (S473) P-Akt (S473) Akt Akt HSP90 HSP90 A 2-DG Aktinh 0 Aktinh 2-DG Sesn2 B C P-Akt (S473) Hsp 90 *p=0.045 ** p= 0.002 ns ns D E Sesn2 mRNA relative expression Sesn2 mRNA relative expression Fig. S5: A,B,C: Cells were treated or not with 20mM 2-DG for 15 hours in presence of the Akt1/2 inhibitor (10µM). D: LNCaP cells were treated with LY294002 (10µM) or transfected with siRNA against Akt1 and Akt2 (E) and incubated with 20mM 2-DG for 8h before RNA preparation. mRNA levels are measured by Real time PCR and results are normalized using RPLP0 as an invariant control. The results were expressed relative to the control condition wich was arbitrary assigned a value of 1.

  7. siSesn2 siCT B A 0 2-DG Tuni 0 2-DG Tuni Tunicamycin 2-DG GRP78 120 0 8 17 24 48 8 17 24 48 h. erk2 MEF Wt GRP78 100 120 MEF Sesn2 -/- sesn2 80 100 erk2 Cellviability % of control 80 60 60 40 40 20 20 0 0 0 2-DG HBSS 0 2-DG HBSS E Viabilityassay Cellcounting Caspase 3 activity (relative activity/min./mg prot.) 2.5 D C Sesn2+/+ Sesn2-/- 2-DG 2-DG 2 C C 24h 48h S 24h 48h S 1.5 Number of cells % of control Pro-Caspase 3 1 Caspase 3 c 0.5 0 0 2-DG 2-DG 0 0 2-DG 2-DG 0 Sesn2+/+ Sesn2-/- Fig. S6: A: LNCaP cells were treated for the indicated time with Tunicamycin (10µg/ml) or 2-DG (20mM) before immunoblotting. B: LNCaP were transfected with siRNASesn2 or siCt for 48h and treated with 2-DG or tunicamycin for 24h before immunoblotting. C: Sesn2 wt and Sesn2 -/- Mefs were treated with 2-DG (20mM) for 24h and 48h or Staurosporin (1µM) before immunoblotting D: Measurement of caspase 3 activity in Mefs treated with 2-DG 20mM for 48h. Cell viability assay performed with XTT assay and cell counting of MEFs labelled with DAPI carried out using Flow cytometry.

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