Supplement fig. 4 Effects of Activin A on osteoblastic differentiation of HPDLC-3O and -3Q

**

Cont

Ca Ca+#1

Ca+#2

Ca+#3

Ca+#4

0

10

20

30

40

50

Are

a (m

m2)

**

AR

VK

#1 #4#3#2

AR

VK

ACVCa (-)

(-)(+) (+) (+) (+) (+)(-)

Cont

Ca Ca+#1

Ca+#2

Ca+#3

Ca+#4

0

20

40

60

80

(C)

AR

VK

Are

a (m

m2)

****

#1 #4#3#2

AR

VK

ACVCa (-)

(-)(+) (+) (+) (+) (+)(-)

AR

VK Cont

Ca Ca+#5

Ca+#6

Ca+#7

Ca+#8

0

20

40

60 ****

Are

a (m

m2)

#5 #8#7#6

AR

VK

ACVCa (-)

(-)(+) (+) (+) (+) (+)(-)

Cont

Ca Ca+#5

Ca+#6

Ca+#7

Ca+#8

0

20

40

60

80

****

Are

a (m

m2)

#5 #8#7#6

AR

VK

ACVCa (-)

(-)(+) (+) (+) (+) (+)(-)

Supplemental Fig. 4 Effects of Activin A on osteoblastic differentiation of HPDLC-3O and -3Q.(A) HPDLC-3O, derived from a premolar of 20-year-old female (upper) and -3Q, derived from a premolar of a 21-year-old female (bottom) were cultured in 10%FBS/MEM as a control, 10%FBS/MEM + 1 mM CaCl2 (Ca), or 10%FBS/MEM + 100 ng/ml Activin A (ACV), or 10%FBS/MEM + 1 mM CaCl2 + 100 ng/ml Activin A (Ca + ACV). Mineralization was visualized by Alizarin Red S staining (AR) and von Kossa staining (VK) on Day 21 and staining area was quantitated. (B) Gene expression changes for OCN, RUNX2 and BSP in HPDLC-3O (upper) and -3Q (bottom) cultured in one of the aforementioned conditions was examined by quantitative RT-PCR analysis. The expression levels of these genes were normalized against -actin expression, and the results are shown as the fold-increase of the control. (C) HPDLC-3O (upper) and -3Q (bottom) were cultured under different timeframes and durations in the presence or absence of Activin A treatment with or without 1 mM CaCl2 for 3 weeks. The schedules of Activin A exposure are described in Fig. 4C. After culturing, mineralization was visualized by AR and VK staining methods on day 21, and positively-stained areas were quantified and described graphically. Values are the mean ± SD from three independent experiments. *P < 0.05 , **P < 0.01. These results support the findings presented in Fig. 4.

(A)* **

(B)

** * *

AR

VK

Cont Ca ACVCa

+ACV

Are

a (m

m2)

Ca

Ca+ACV

0

10

20

30 **

AR

VK

Cont Ca ACVCa

+ACV

Ca

Ca+ACV

0

10

20

30

40 **A

rea

(mm

2)

mR

NA

exp

ress

ion

(/b

-act

in)

mR

NA

exp

ress

ion

(/b

-act

in)

Cont

CaACV

Ca+ACV

01234

OCN

Cont

CaACV

Ca+ACV

02468

RUNX2

Cont

CaACV

Ca+ACV

0

1

2

3

4

BSP

Cont

CaACV

Ca+ACV

05

10152025

OCN

Cont

CaACV

Ca+ACV

0

1

2

3

4

RUNX2

Cont

CaACV

Ca+ACV

0

2

4

6

BSP