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
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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).
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Figure S1
(A)
(B)
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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.
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5
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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
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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.
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3
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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
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5
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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).
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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
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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.
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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
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4
5
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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
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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.
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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
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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)
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