Supplemental methods

RNA preparation and quantitative real-time PCR

Total RNA of treated cells was isolated by TRIzol (Invitrogen). RNA samples were

treated with the RQ1 RNase-free DNase (Promega) to remove any genomic

contamination according to the manufacturer’s instructions. Five micrograms of

treated RNA samples was subjected to reverse transcription with SuperScript III

(Invitrogen). Quantitative real-time PCR was processed by StepOne Real-Time PCR

System (Applied Biosystems) using Maxima Hot Start PCR Master Mix (2)

(Fermantus), and GAPDH was used as an internal control. Besides melting curve, real

time PCR products were also analyzed by gel electrophoresis to confirm single PCR

products. Primer sets were listed in supplemental table 1.

Flow cytometry

Subconfluent cells were trypsinized, washed with PBS. A total of 1 x106 cells were

fixed with 100% ethanol for 10 min following incubated with 1 mg/ml propidium

iodide (PI) for 10 min at room temperature. Cells were analyzed within 20 min post-

staining on a BD FACSCalibur (BD Biosciences). Quantitation of different cell cycle

phases were categorized by PI-staining intensity as sub-G1 (<800), G1 (800-1200), S

(1201-1700), and G2/M (1701-2100). For Annexin V-PI double staining, treated cells

1

12

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

12

were harvested at indicated time, wash twice with ice cold PBS, and directly stained

with FITC-conjugated Annexin V and PI (BD Biosciences) for 15 min without

fixation. Quadrant was divided as x-axis: Annexin-positive (FITC intensity >=45) and

Annexin-negative (FITC intensity <45), and y-axis: PI-positive (PI intensisty >=40)

and PI-negative (PI intensity <40).

2

1

2

3

4

5

6

12

Figure S1

(A)

(B)

3

1

2

3

4

5

6

7

8

12

Figure S1. Isolation of H. syriacus skin extracts

Root skin powder from H. syriacus (8.70 kg) was rinsed three times with non-polar

organic solvent acetone, and the acetone layer was obtained. Following by increasing

the polarity of organic solvent to isolate acetone extract, and the crude extracts HISY-

F1 (Hibiscus syriacus), HISY-F2, HISY-F3, HISY-F4, HISY-F5, HISY-F6 and HISY-

F7. Subsequently, the polarity of organic solvent increased again, and the pure

compounds K01-K14 were extracted from HISY-F2, HISY-F4, HISY-F5 and HISY-

F6.

4

1

2

3

4

5

6

7

8

9

10

12

Figure S2

PI K04 24.003

FL2-H

Cou

nt

100 101 102 103 1040

59

117

176

234

M1

M2M3

PI N.C 48.010

FL2-H

Cou

nt

100 101 102 103 1040

15

30

45

60

M1

M2

M3

PI V.C 48.009

FL2-H

Cou

nt

100 101 102 103 1040

17

34

50

67

M1

M2

M3

PI K02 48.005

FL2-H

Cou

nt

100 101 102 103 1040

23

46

69

92

M1

M2

M3

PI N.C 48.012

FL2-H

Cou

nt

100 101 102 103 1040

14

29

43

57

M1

M2

M3

DMSO (0.1% DMSO)

K02 (10µg/ml)

K03 (10µg/ml)

K04 (10µg/ml)

K06 (10µg/ml)

Sub-G1

G1

S

G2/M

Sub-G1

G1

S

G2/M

Sub-G1

G1

S

G2/M

Sub-G1

G1

S

G2/M

Sub-G1

G1

S

G2/M

A

B

C

D

E

0%

20%

40%

60%

80%

100%

DMSO K02 K03 K04 K06

Perc

enta

ge (/

tota

l)

Sub-G1

G1

S

G2/M

** * *

**

Figure S2. Betulin and its derivatives increased the sub-G1 population of HBL-100

cells

5

1

2

3

4

5

12

After treating HBL-100 with 0.1% DMSO (A), or 10 µg/mL of K02 (B), K03 (C),

K04 (D) or K06 (E) for 48 h, the cells were stained with PI for flow cytometry.

Compared to DMSO control (A), HBL-100 cells treated with K02, K03, K04, and

K06 showed a significant increase in sub-G1 population, in particular the cells treated

with K02 (B) and K06 (E). Quantitation of cells in sub-G1, G1, and G2/M phases

showed that over 50% of cells were in sub-G1 phase in cells treated with K02 and

K06.

6

1

2

3

4

5

6

7

8

12

Figure S3

AP K02 48.013

FL1-H

FL2-

H

100 101 102 103 104100

101

102

103

1048.91% 12.84%

51.67% 26.58%

AP K06 48.016

FL1-H

FL2-

H

100 101 102 103 104100

101

102

103

1049.57% 19.02%

32.54% 38.87%

AP V.C 48.017

FL1-H

FL2-

H

100 101 102 103 104100

101

102

103

1048.58% 7.23%

69.96% 14.23%

AP N.C 48.018

FL1-H

FL2-

H

100 101 102 103 104100

101

102

103

1048.63% 7.52%

68.86% 14.99%

K02 (10μg/ml) K03 (10μg/ml)

K04 (10μg/ml) K06 (10μg/ml)

AP K04 48.015

FL1-H

FL2-

H

100 101 102 103 104100

101

102

103

104

12.10% 1.38%

85.84% 0.68%

HBL-100DMSO (0.1%)

AnnexinV

PI AnnexinVPI

AnnexinV

PI

AnnexinV

PI

AnnexinV

PI

A10.21%

87.73%

B C

D E

87.73

51.67

69.96 68.86

32.54

0.68

26.58

14.23 14.99

38.87

1.3812.84 7.23 7.52

19.02

10.21 8.91 8.58 8.63 9.57

0

10

20

30

40

50

60

70

80

90

100

DMSO K02 K03 K04 K06

Perc

enta

ge other

late

earily

survival

F

Figure S3. Betulin and its derivatives induced apoptosis in HBL-100 cells.

After treating HBL-100 with 10 µg/mL of K02 (B), K03 (C), K04 (D) and K06 (E)

for 48 h, the cells were double stained with FITC-conjugated Annexin V and PI for

7

1

2

3

4

5

6

7

12

flow cytometry. The results were quantified for each quadrant. Compared to DMSO

control (0.1% DMSO) (A), cells in quadrant I and IV increased in cells treated with

K02, K03, K04 and K06, especially in cells treated with K02 (B) and K06 (E). After

quantifying all four quadrants, cell number in quadrant I and IV from K02 and K06

treated groups was more remarkably increased whilst compared to those of K03 and

K04 treated and DMSO control groups (F).

8

1

2

3

4

5

6

7

12

Figure S4.

0.1

1

10

1000h

r12

hr24

hr36

hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr

TAp63 ΔNp63 BAX NOXA PUMA PERP

Rela

tive m

RNA e

xpre

ssion

(nor

mali

zed t

o GAD

PH)

K02

0.1

1

10

100

0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr

TAp63 ΔNp63 BAX NOXA PUMA PERP

Relat

ive m

RNA

expr

essio

n (no

rmali

zed t

o GA

DPH)

K06

0.1

1

10

100

0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr

TAp63 ΔNp63 BAX NOXA PUMA PERP

Rel

ative

mRN

A ex

pres

sion (

norm

alize

d to G

ADPH

)

K03

0.1

1

10

100

0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr

TAp63 ΔNp63 BAX NOXA PUMA PERP

Rela

tive m

RNA e

xpre

ssion

(nor

mali

zed t

o GA

DPH)

K04

A D

CB

****

**

**** **

* **

*

**

*

****

***

***

***

***

**

**** **

**

*

****

**

**

***

**

***

**

Figure S4. Betulin and its derivatives regulated apoptotic-related gene mRNA

expression in MDA-MB-231 cells.

After treating MDA-MB-231 with 10 µg/mL of K02 (A), K03 (B), K04 (C) or K06

(D) for 0, 12, 24 and 36 h, real-time PCR was performed to analyze TAp63, ΔNp63,

BAX, PUMA, NOXA and PERP mRNA expression. Among K02 and K06 treated

cells, TAp63 was significantly increased at 12 h and slightly decreased at 24 h and 36

h. ΔNp63 also increased at 12 h, and it lost significance and dramatically decreased at

24 h or 36 h. And most of TAp63 downstream apoptotic genes including BAX,

NOXA, PUMA and PERP were increased over time (A and D). These results

9

1

2

3

4

5

6

7

8

9

10

11

12

implicated that K02 and K06 induced apoptotic gene expression in a TAp63-

associated manner. Although K03 and K04 treatment also increased TAp63

expression, no significant change was observed in ΔNp63 or downstream apoptotic

genes (B and C). Therefore, in p53-mutated MDA-MB-231 breast cancer cells, K02

and K06 may induce TAp63 expression to compensate parts of p53 function.

10

1

2

3

4

5

6

12

Figure S5.

0.1

1

10

1000h

r12

hr24

hr36

hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr

TAp63 ΔNp63 BAX NOXA PUMA PERP

Rela

tive

mRN

A ex

pres

sion

(nor

mal

ized

to G

ADPH

) K02

0.1

1

10

100

0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr

TAp63 ΔNp63 BAX NOXA PUMA PERP

Rela

tive

mRN

A ex

pres

sion

(nor

mal

ized

to G

ADPH

)

K06

0.1

1

10

100

0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr

TAp63 ΔNp63 BAX NOXA PUMA PERP

Rela

tive

mRN

A ex

pres

sion

(nor

mal

ized

to G

ADPH

)

K03

0.1

1

10

100

0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr 0hr

12hr

24hr

36hr

TAp63 ΔNp63 BAX NOXA PUMA PERP

Rela

tive

mRN

A ex

pres

sion

(nor

mal

ized

to G

ADPH

)

K04CB

A D

***

**

* **

*

* ** *

(E) MTT assay

0%

20%

40%

60%

80%

100%

120%

0 0.1 1 2.5 5 10

Cell

viab

ility

(ref

er to

DM

SO)

μg/ml

pure compound

DMSO

K02

K03

K04

K06

(F) Flow cytometry

11

1

2

3

4

5

67

12

A. DMSO (0.1% DMSO) B. K02 (10µg/ml)

C. K03 (10µg/ml) D. K04 (10µg/ml) E. K06 (10µg/ml)

Figure S5. The effects of betulin and its derivatives on cell viability, apoptosis and

apoptotic-related gene expression in non-tumorigenic human breast epithelial cell

H184B5F5/M10.

After treating H184B5F5/M10 with 10 µg/mL of K02 (A), K03 (B), K04 (C) or K06

(D) for 0, 12, 24 and 36 h, real-time PCR was performed to analyze TAp63, ΔNp63,

BAX, PUMA, NOXA and PERP mRNA expression. Among all treated cells, the

expression of BAX, NOXA, PUMA and PERP was not changed. In, TAp63

expression increased first and decreased afterwards in K02 or K06 treated cells.

ΔNp63 increased first and decreased afterwards in K02 treated cells and fluctuated in

K06 treated cells. In K03 and K04 treated cells, ΔNp63 was not obviously changed,

and the expression of TAp63 increased first and decreased over time. Besides,

H184B5F5/M10 cells were treated with 0.1% DMSO, or 10 µg/mL of K02, K03, K04

or K06 for 48 h and harvested for (E) MTT assay and (F) PI-staining flow cytometry.

The detail methods were identical as described in text, legend of Figure 1, and

12

12

3

4

5

6

7

8

9

10

11

12

13

14

15

16

12

supplemental methods. Betulin and its derivatives were not obviously modified cell

viability and apoptosis of H184B5F5/M10 cells and therefore implicated a relative

lower toxicity to normal mammary cells.

13

1

2

3

4

12

Supplemental table 1 Primer list of real time PCR

Forward primer Reverse primer

TAp63 CAGTCCAGAGGTTTTCCAGCAT TCAATGGGCTGAACATATAG

ΔNp63 GCAAAACAATGCCCAGACTCA TGTTCAGGAGCCCCAGGTT

BAX ATGTTTTCTGACGGCAACTTC ATCAGTTCCGGCACCTTG

PUMA ACCTCAACGCACAGTACGA GAGATTGTACAGGACCCTCCA

NOXA GGAGATGCCTGGGAAGAAG CCTGAGTTGAGTAGCACACTCG

PERP TGTCTTCCTGAGAGTGATTGGA ACCAGGGAGATGATCTGGAA

GAPDH CCACTCCTCCACCTTTGAC ACCCTGTTGCTGTAGCCA

14

1

2

12

Supplemental table 2 Antibodies listPrimary Antibody Secondary Antibody

Bax Rabbit polyclonal Ab(Santa cruz)

HRP-conjugated Goat anti-rabbit IgG Ab

(Jackson)Bcl-x Rabbit polyclonal Ab

(Santa cruz)HRP-conjugated Goat

anti-rabbit IgG Ab(Jackson)

Caspase-3 Rabbit polyclonal Ab(Cell signaling)

HRP-conjugated Goat anti-rabbit IgG Ab

(Jackson)PARP Rabbit polyclonal Ab

(Cell signaling)HRP-conjugated Goat

anti-rabbit IgG Ab(Jackson)

p53 Mouse monoclonal Ab(Dako)

HRP-conjugated Goat anti-mouse IgG Ab

(Jackson)p21 Mouse monoclonal Ab

(Santa cruz)HRP-conjugated Goat

anti-mouse IgG Ab(Jackson)

Phospho-AKT

(Ser473)

Rabbit polyclonal Ab(Cell signaling)

HRP-conjugated Goat anti-rabbit IgG Ab

(Jackson)

Pan-AKT Rabbit polyclonal Ab(Cell signaling)

HRP-conjugated Goat anti-rabbit IgG Ab

(Jackson)β-actin Mouse monoclonal Ab

(Thermo SCIENTIFIC)HRP-conjugated Goat

anti-mouse IgG Ab(Jackson)

15

1

2

12