1
2
Supplemental methods
3
RNA preparation and quantitative real-time PCR
4
Total RNA of treated cells was isolated by TRIzol (Invitrogen). RNA samples were
5
treated with the RQ1 RNase-free DNase (Promega) to remove any genomic
6
contamination according to the manufacturer’s instructions. Five micrograms of
7
treated RNA samples was subjected to reverse transcription with SuperScript III
8
(Invitrogen). Quantitative real-time PCR was processed by StepOne Real-Time PCR
9
System (Applied Biosystems) using Maxima Hot Start PCR Master Mix (2)
10
(Fermantus) and GAPDH was used as an internal control. Besides melting curve, real
11
time PCR products were also analyzed by gel electrophoresis to confirm single PCR
12
products. Primer sets were listed in supplemental table 1.
13
14
Flow cytometry
15
Subconfluent cells were trypsinized, washed with PBS. A total of 1 x106 cells were
16
fixed with 100% ethanol for 10 min following incubated with 1 mg/ml propidium
17
iodide (PI) for 10 min at room temperature. Cells were analyzed within 20 min
18
post-staining on a BD FACSCalibur (BD Biosciences). Quantitation of different cell
19
cycle phases were categorized by PI-staining intensity as sub-G1 (<800), G1
20
(800-1200), S (1201-1700), and G2/M (1701-2100). For Annexin V-PI double
1
1
staining, treated cells were harvested at indicated time, wash twice with ice cold PBS,
2
and directly stained with FITC-conjugated Annexin V and PI (BD Biosciences) for 15
3
min without fixation. Quadrant was divided as x-axis: Annexin-positive (FITC
4
intensity >=45) and Annexin-negative (FITC intensity <45), and y-axis: PI-positive
5
(PI intensisty >=40) and PI-negative (PI intensity <40).
6
2
1
S Fig 1.
2
(A)
3
4
5
(B)
6
7
8
9
3
1
S Fig 1. Isolation of H. syriacus skin extracts
2
Root skin powder from H. syriacus (8.70 kg) was rinsed three times with non-polar
3
organic solvent acetone, and the acetone layer was obtained. Following by increasing
4
the polarity of organic solvent to isolate acetone extract, and the crude extracts
5
HISY-F1 (Hibiscus syriacus), HISY-F2, HISY-F3, HISY-F4, HISY-F5, HISY-F6 and
6
HISY-F7. Subsequently, the polarity of organic solvent increased again, and the pure
7
compounds K01-K14 were extracted from HISY-F2, HISY-F4, HISY-F5 and
8
HISY-F6.
9
4
1
S Fig 2.
K03 (10µg/ml)
PI V.C 48.009
67
C
50
Count
DMSO (0.1% DMSO)
PI K04 24.003
234
M1
Sub-G1
34
A
176
Count
G1
M2
17
S
Sub-G1
M1
117
M3
G2/M
0
0
G1M2
1
10
G2/M
M3
2
10
10
3
4
10
10
FL2-H
K04 (10µg/ml)
59
PI K02 48.005
92
D
S
0
0
1
10
10
2
3
10
4
10
10
69
Count
FL2-H
K02 (10µg/ml)
M1
Sub-G1
46
M2
G1
23
M3
G2/M
PI N.C 48.010
S
60
B
0
0
1
10
2
10
10
4
10
FL2-H
45
Count
3
10
K06 (10µg/ml)
Sub-G1
M1
30
PI N.C 48.012
G1
M2
57
G2/M
M3
E
15
43
Count
S
0
0
1
10
10
2
3
10
Sub-G1
29
M1
4
10
10
G1
FL2-H
G2/M
M3
M2
14
S
0
0
10
2
1
10
2
10
3
10
4
10
FL2-H
**
80%
**
Percentage (/total)
100%
60%
*
*
Sub-G1
G1
40%
S
G2/M
20%
0%
DMSO
K02
K03
K04
K06
3
4
S Fig 2. Betulin and its derivatives increased the sub-G1 population of HBL-100 cells
5
After treating HBL-100 with 0.1% DMSO (A), or 10 µg/mL of K02 (B), K03 (C),
6
K04 (D) or K06 (E) for 48 h, the cells were stained with PI for flow cytometry.
5
1
Compared to DMSO control (A), HBL-100 cells treated with K02, K03, K04, and
2
K06 showed a significant increase in sub-G1 population, in particular the cells treated
3
with K02 (B) and K06 (E). Quantitation of cells in sub-G1, G1, and G2/M phases
4
showed that over 50% of cells were in sub-G1 phase in cells treated with K02 and
5
K06.
6
6
1
S Fig 3.
B
10
10
10
12.84%
3
10
2
12.10%
10.21%
PI
FL2-H
7.23%
3
2
1
10
1
3
51.67%
26.58%
69.96%
0
10
8.58%
1.38%
10
0
10
10
10
4
4
10
10
K03
(10μg/ml)
AP V.C
48.017
C
8.91%
PI
FL2-H
HBL-100
DMSO
AP K04 (0.1%)
48.015
A
PI
10
K02
(10μg/ml)
AP K02
48.013
4
FL2-H
10
1
D
10
85.84%
87.73%
10
2
FL1-H
Annexin
V
2
1
14.23%
0
10
10
3
10
10
0
10
4
10
E
K04
(10μg/ml)
AP N.C
48.018
4
8.63%
10
7.52%
1
10
2
FL1-H
Annexin
V
10
3
10
4
K06
(10μg/ml)
AP K06
48.016
4
9.57%
19.02%
0.68%
0
2
10
3
10
4
10
10
10
3
10
2
1
68.86%
2
90
10.21
80
8.91
1.38
0.68 12.84
Percentage
70
2
1
32.54%
38.87%
0
10
1
10
2
FL1-H
Annexin
V
10
3
10
4
8.58
8.63
7.23
7.52
14.23
14.99
10
0
10
10
1
10
2
FL1-H
Annexin
V
10
3
10
4
9.57
19.02
26.58
60
other
38.87
50
40
3
14.99%
0
100
10
10
10
0
10
F
PI
10
FL1-H
Annexin
V
FL2-H
1
PI
10
FL2-H
10 0
10
87.73
69.96
30
earily
68.86
survival
51.67
20
late
32.54
10
0
DMSO
K02
K03
K04
K06
3
4
5
S Fig 3. Betulin and its derivatives induced apoptosis in HBL-100 cells.
6
After treating HBL-100 with 10 µg/mL of K02 (B), K03 (C), K04 (D) and K06 (E)
7
for 48 h, the cells were double stained with FITC-conjugated Annexin V and PI for
7
1
flow cytometry. The results were quantified for each quadrant. Compared to DMSO
2
control (0.1% DMSO) (A), cells in quadrant I and IV increased in cells treated with
3
K02, K03, K04 and K06, especially in cells treated with K02 (B) and K06 (E). After
4
quantifying all four quadrants, cell number in quadrant I and IV from K02 and K06
5
treated groups was more remarkably increased whilst compared to those of K03 and
6
K04 treated and DMSO control groups (F).
7
8
S Fig 4.
PUMA
**
**
**
**
**
**
**
12hr
**
**
36hr
**
PERP
BAX
NOXA
PUMA
36hr
24hr
12hr
0hr
36hr
24hr
0hr
24hr
12hr
0hr
36hr
24hr
0hr
ΔNp63
12hr
36hr
24hr
0hr
TAp63
PERP
0.1
TAp63
ΔNp63
BAX
NOXA
PUMA
**
K04
PERP
TAp63
0.1
ΔNp63
BAX
NOXA
PUMA
36hr
0hr
24hr
12hr
36hr
24hr
12hr
0hr
36hr
24hr
12hr
0hr
36hr
24hr
12hr
0hr
36hr
0hr
24hr
12hr
36hr
1
0hr
36hr
24hr
12hr
0hr
36hr
24hr
12hr
0hr
36hr
0hr
24hr
12hr
36hr
24hr
12hr
0hr
36hr
24hr
0hr
12hr
36hr
0hr
24hr
12hr
1
10
24hr
**
10
100
K03
Relative mRNA expression (normalized to GADPH)
100
***
C
***
B
12hr
0.1
Relative mRNA expression (normalized to GADPH)
*
1
12hr
36hr
24hr
12hr
0hr
36hr
24hr
0hr
NOXA
12hr
36hr
24hr
0hr
BAX
12hr
36hr
24hr
0hr
ΔNp63
12hr
36hr
0hr
24hr
12hr
36hr
0hr
24hr
1
*
36hr
*
*
K06
10
0hr
*
100
24hr
*
**
**
*
**
**
**
**
**
**
**
10
12hr
Relative mRNA expression (normalized to GADPH)
K02
TAp63
2
***
***
***
D
100
12hr
A
Relative mRNA expression (normalized to GADPH)
1
PERP
0.1
3
S Fig 4. Betulin and its derivatives regulated apoptotic-related gene mRNA expression
4
in MDA-MB-231 cells.
5
After treating MDA-MB-231 with 10 µg/mL of K02 (A), K03 (B), K04 (C) or K06
6
(D) for 0, 12, 24 and 36 h, real-time PCR was performed to analyze TAp63, ΔNp63,
7
BAX, PUMA, NOXA and PERP mRNA expression. Among K02 and K06 treated
8
cells, TAp63 was significantly increased at 12 h and slightly decreased at 24 h and 36
9
h. ΔNp63 also increased at 12 h, and it lost significance and dramatically decreased at
10
24 h or 36 h. And most of TAp63 downstream apoptotic genes including BAX,
11
NOXA, PUMA and PERP were increased over time (A and D). These results
9
1
implicated that K02 and K06 induced apoptotic gene expression in a
2
TAp63-associated manner. Although K03 and K04 treatment also increased TAp63
3
expression, no significant change was observed in ΔNp63 or downstream apoptotic
4
genes (B and C). Therefore, in p53-mutated MDA-MB-231 breast cancer cells, K02
5
and K06 may induce TAp63 expression to compensate parts of p53 function.
6
10
Cell viability (refer to DMSO)
2
4
6
7
TAp63
0
ΔNp63
BAX
0.1
5
*
NOXA
1
PUMA
2.5
PERP
TAp63
K03
10
*
1
0.1
60%
40%
20%
0%
5
10
μg/ml
(F) Flow cytometry
11
ΔNp63
C
100
0.1
TAp63
K04
K06
ΔNp63
3
(E) MTT assay
pure compound
120%
DMSO
100%
K02
80%
K03
BAX
BAX
NOXA
NOXA
PUMA
PUMA
0hr
12hr
24hr
36hr
PERP
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
*
0hr
12hr
24hr
36hr
B
*
0hr
12hr
24hr
36hr
0.1
0hr
12hr
24hr
36hr
D
100
0hr
12hr
24hr
36hr
1
0hr
12hr
24hr
36hr
*
Relative mRNA expression (normalized to GADPH)
K02
0hr
12hr
24hr
36hr
PUMA
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
A
10
Relative mRNA expression (normalized to GADPH)
100
NOXA
0hr
12hr
24hr
36hr
BAX
0hr
12hr
24hr
36hr
ΔNp63
0hr
12hr
24hr
36hr
100
0hr
12hr
24hr
36hr
TAp63
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
*
**
*
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
Relative mRNA expression (normalized to GADPH)
10
0hr
12hr
24hr
36hr
Relative mRNA expression (normalized to GADPH)
1
S Fig 5.
K06
10
*
*
1
0.1
PERP
K04
**
1
PERP
A. DMSO (0.1% DMSO)
C. K03 (10µg/ml)
B. K02 (10µg/ml)
D. K04 (10µg/ml)
E. K06 (10µg/ml)
1
2
3
S Fig 5. The effects of betulin and its derivatives on cell viability, apoptosis and
4
apoptotic-related gene expression in non-tumorigenic human breast epithelial cell
5
H184B5F5/M10.
6
After treating H184B5F5/M10 with 10 µg/mL of K02 (A), K03 (B), K04 (C) or K06
7
(D) for 0, 12, 24 and 36 h, real-time PCR was performed to analyze TAp63, ΔNp63,
8
BAX, PUMA, NOXA and PERP mRNA expression. Among all treated cells, the
9
expression of BAX, NOXA, PUMA and PERP was not changed. In, TAp63
10
expression increased first and decreased afterwards in K02 or K06 treated cells.
11
ΔNp63 increased first and decreased afterwards in K02 treated cells and fluctuated in
12
K06 treated cells. In K03 and K04 treated cells, ΔNp63 was not obviously changed,
13
and the expression of TAp63 increased first and decreased over time. Besides,
14
H184B5F5/M10 cells were treated with 0.1% DMSO, or 10 µg/mL of K02, K03, K04
15
or K06 for 48 h and harvested for (E) MTT assay and (F) PI-staining flow cytometry.
16
The detail methods were identical as described in text, legend of figure 1, and
12
1
supplemental methods. Betulin and its derivatives were not obviously modified cell
2
viability and apoptosis of H184B5F5/M10 cells and therefore implicated a relative
3
lower toxicity to normal mammary cells.
4
13
1
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
2
14
1
Supplemental table 2 Antibodies list
Primary Antibody
Secondary Antibody
Bax
Rabbit polyclonal Ab
(Santa cruz)
Bcl-x
Rabbit polyclonal Ab
(Santa cruz)
Caspase-3
Rabbit polyclonal Ab
(Cell signaling)
PARP
Rabbit polyclonal Ab
(Cell signaling)
p53
Mouse monoclonal Ab
(Dako)
p21
Mouse monoclonal Ab
(Santa cruz)
Phospho-AKT
Rabbit polyclonal Ab
(Cell signaling)
HRP-conjugated Goat
anti-rabbit IgG Ab
(Jackson)
HRP-conjugated Goat
anti-rabbit IgG Ab
(Jackson)
HRP-conjugated Goat
anti-rabbit IgG Ab
(Jackson)
HRP-conjugated Goat
anti-rabbit IgG Ab
(Jackson)
HRP-conjugated Goat
anti-mouse IgG Ab
(Jackson)
HRP-conjugated Goat
anti-mouse IgG Ab
(Jackson)
HRP-conjugated Goat
anti-rabbit IgG Ab
(Jackson)
(Ser473)
Pan-AKT
Rabbit polyclonal Ab
(Cell signaling)
β-actin
Mouse monoclonal Ab
(Thermo SCIENTIFIC)
2
15
HRP-conjugated Goat
anti-rabbit IgG Ab
(Jackson)
HRP-conjugated Goat
anti-mouse IgG Ab
(Jackson)