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).

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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).

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.


Suplementary experimental procedures

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


Suplementary experimental procedures

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.


Suplementary experimental procedures

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.


Suplementary experimental procedures

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.


Suplementary experimental procedures

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.


Suplementary experimental procedures

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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