Results

STARTED : 2016-04
Osteoblast Differentiation

E1 E2

Differentiation Media - + - +

Differentiation Media - +
Id1

Dlx5

Runx2

b-actin

• MC3T3-E1 (8*10^4)
• MC3T3-E1 (3*10^4)
• Incubate 2 day
• Incubate 2 day
• Differentiation 4 day • Differentiation 4 day
• Ascorbic acid • Ascorbic acid
• b-glycerphosphatate • b-glycerphosphatate

FIG: DAY-5, ALP staining (Early Differentiation Marker)

 Differentiation Media - +

E1 E2

- + - +

ALP

OC

b-actin

• MC3T3-E1 (8*10^4) • MC3T3-E1 (8*10^4)

• Incubate 2 day • Incubate 2 day
• Differentiation 8 day • Differentiation 8 day
• Ascorbic acid • Ascorbic acid
• b-glycerphosphatate • b-glycerphosphatate

FIG: DAY-9, Alzarian-Red stain (Late differentiation marker)

 ALP Staining

- +

• MC3T3-E1 (3*10^4)
• Incubate 2 day
• Differentiation 4 day
• Ascorbic acid
• b-glycerphosphatate
MTT Result with Suramin Concentration

0.03
0.26

0.04
0.0008
0.0005
 ALP staining – Suramin Concentration

Control 10 uM 20 uM 50 uM 100 uM

• MC3T3-E1 (1.5*10^5)
Incubate 2 day
• Differentiation 4 day
• Ascorbic acid (50ug/ml)
• b-glycerphosphatate (5mM)

200 uM 400 uM
*

MTT assay

Alp staining
 ALP staining – 50uM Suramin Concentration
8 Days

• MC3T3-E1 (1.5*10^5)
• Differentiation 8 days
• Ascorbic acid (50ug/ml) Differentiation Media Differentiation Media With
• b-glycerphosphatate Without Suramin Suramin
(5mM)

ARS staining – 50uM Suramin

Concentration 6 Days
• MC3T3-E1 (1.5*10^5)
• Ascorbic acid (50ug/ml)
• b-glycerphosphatate
(5mM)
Differentiation Media Differentiation Media
Without Suramin With Suramin
 mRNA expression of Osteoblast differentiataion markers With and
without suramin
25

20
mRNA expression

15
RUNX-2
ALP
10 DLX-5
OC

0
0 2days - 2 days 4 days 4 days 8 days- 8days+
-= Differentiation media
+ - + += Differentiation media +
Days
suramin
ARS staining – 50uM Suramin Concentration
21 Days

Differentiation Media Differentiation Media

Without Suramin With Suramin
• MC3T3-E1 (1.5*10^5)
• Differentiation 21 days
• Ascorbic acid (50ug/ml)
• b-glycerphosphatate (5mM)
 Western Results
Growth media vs differentiation media 6 hour after suramin treatment

Medium Growth Differentiation

Su (50mM) + +
P-smad1/5/8

T-smad1/5/8

PDK 4

PDK 1

GAPDH

P- AKT

T- AKT

P- pi3K
 Western Results
Growth media vs differentiation media 6 hour after suramin treatment
GM GM + Su DM DM+Su

P-
smad1/5/8

T-
smad1/5/8

P- AKT

T- AKT

P- ERK

P- pi3K

PDK 4

PDK 1

GAPDH
 Western Results
Differentiation media 2 days. Time dependent after suramin treatment 15m-2 hr
15 m 30 m 1 hour 2 hour

C - + - + - + - +
GAPDH

P -ERK

P Smad

P -ERK

T-ERK

P AKT

T AKT
 ARS staining – 50uM Suramin Concentration
21 Days

Differentiation Media Differentiation Media

Without Suramin With Suramin Differentiation Media Differentiation Media
Without Suramin With Suramin
 BardFord Assay
12

10

0
2 ug 4ug 6ug 8ug 10ug
mRNA expression of Runx2 and ALP with 50mM of suramin for 8 days in differentiation
media.

3.50

3.00

2.50

2.00

Runx2 ALP

1.50

1.00

0.50

0.00
0 2C 2T 4C 4T 8C 8T
 ALP staining 4 days Growth media , Differential Media +
50uM Suramin.

- - +
+
GM

DM
 ARS staining 21 days Differential Media + 50uM
Suramin.

DM

DM +
Suramin

14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
Control Treat
 14
DM 12

10

DM + Su 2

0
Control Treat

E: Alizarin Red staining F: Alizarin Red staining

Quantification
mRNA expression of Osteoblast differentiataion markers With and
without suramin
PDK mRNA expression in Osteoblast differentiation markers With
and without suramin
 Western Results
Osteoblast differentiation with or without suramin (50uM) 0-8 days
Days 0 2 4 8
Su (50uM) - - + - + - +
PDK 1
P-Smad 1/5/8
P-PI3K
mTOR
Rictor
PDK 3
HK 2
LDHA
OPN
BMP2

Hif1 a
GAPDH
PDK 2
 Western Results
Osteoblast differentiation with or without suramin (50uM
2 days DM, then time dependent upto 6 hr.

Time 0 30m 1hr 2 hr 3hr 6hr

Su (50uM) - - + - + - + - + - +
P-Smad 1/5/8

Hif 1 α

P - AKT

GAPDH
 Osteoblast differentiation with or without suramin (50uM) in Growth media

Time 0 30m 1hr 2 hr 3hr 6hr

Su (50uM) - - + - + - + - + - +
P-Smad 1/5/8
P β- catenin
 Osteoblast differentiation with suramin co treated with DCA

DM + + + + + +
Suramin - + + + + +
DCA (mM) - - 0.5 1 5 10
 Osteoblast differentiation with suramin co treated with DCA

DM + + + + + +
Suramin - + + + + +
DCA (mM) - - 0.5 1 5 10

DM + + + +
Suramin - + + +
DCA (mM) - - 0.5 1
6 hr 12 hr
PTH Promotes Bone Anabolism by Stimulating
Aerobic Glycolysis via IGF Signaling

doi:10.1002/jbmr.2556.
WNT-LRP5 Signaling Induces Warburg Effect through mTORC2 Activation during Osteoblast Differentiation
Suggestive Hypothesis Suramin
B
M
P2

pi3k
Smad1/5/8
mtor 2

Smad 1/5/8 PDK

Anaerobic Glycolysis

DLX5 Runx2 OC
ID1 ALP