Suplementary experimental procedures:
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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 2.10 3 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.
description
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). - PowerPoint PPT Presentation
Transcript of 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).
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.
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.
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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.
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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.
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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.
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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.
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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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