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 (2X)
(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 table 1.
Western blot analysis
Treated cells were washed twice with PBS, then lyzed in 200μl of RIPA lysis buffer
(Millipore, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1%
sodium deoxycholate, 0.1% SDS) containing protease inhibitor (Roche). 30μg of
protein from the supernatant was loaded on SDS polyacrylamide gel followed by
western blot analysis to detect the protein level of indicated genes (antibody
information was listed in Table 2). The immuno-reactive bands were revealed by ECL
system (Millipore) then developed and quantified on UVP BioSpectrum Imaging
System.
Flow cytometry
Subconfluent cells were trypsinized, washed in 1xPBS and adjusted to 2x106 cells⁄ml.
A total of 1 x106 cells were fixed with 100% EtOH for 10 min following incubated
with 1mg/ml propidium iodide for 10 min at room temperature. Cells were analyzed
within 20min post-staining on a BD FACSCalibur (BD Biosciences). And for Annexin
V-PI double staining treated cells were harvested in indicated time, wash twice with
ice cold 1x PBS, and directly stained with Annexin V and propidium iodide for 15
min without fixation as manuscript (BD,Biosciences).
Data analysis
Real-time PCR original data and western blot data were also recorded as
continuous variants and analyzed with Student’s t test or linear polynomial ANOVA
with LSD posthoc examination. All the statistical analyses were performed using
SPSS 16.0 and Excel 2007. All statistical tests and P values were two-sided and the
level of significance was set at < 0.05 (*), < 0.01 (**), or < 0.001 (***).
Supplemental figure 1
(A)
(B)
Supplemental Figure 1. 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.
Supplemental figure 2
K03 (10µg/ml)
PI V.C 48.009
67
C
50
Count
Control (HBL100 48hr)
PI K04 24.003
234
M1
34
M3
A
Count
176
M2
17
0
M1
117
0
1
10
2
10
3
10
4
10
10
FL2-H
M3
K04 (10µg/ml)
M2
59
PI K02 48.005
92
D
0
0
1
10
2
10
10
3
4
10
10
69
Count
FL2-H
M1
46
M3
M2
K02 (10µg/ml)
23
PI N.C 48.010
60
B
0
0
1
10
2
10
3
10
4
10
10
FL2-H
K06 (10µg/ml)
PI N.C 48.012
M1
30
57
M3
E
M2
15
43
Count
Count
45
0
0
10
1
10
2
10
3
10
29
M1
4
10
M3
FL2-H
M2
14
0
0
10
1
10
2
10
3
10
4
10
FL2-H
Supplemental figure 2. Betulin and its derivatives increased the number of HBL-100
cells in sub-G1 phaseAfter treating HBL-100 with 10 µg/mL of K02 (B), K03 (C),
K04 (D) and K06 (E) for 48 hours, the cells were stained with PI for flow cytometry.
Compared to control (untreated HBL-100) (A), HBL-100 cells treated with K02, K03,
K04, and K06 showed a significant increase in sub-G1 phase, 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.
Supplemental figure 3
B
A
10
Control (HBL100 48hr)
FL2-H
10
10
AP K02 48.013
8.91%
10
12.84%
3
10
2
10
K03 (10µg/ml)
AP V.C 48.017
4
8.58%
7.23%
3
2
AP K04 48.015
4
12.10%
1.38%
10
1
10
51.67%
10
C
K02 (10µg/ml)
4
FL2-H
10
69.96%
0
3
1
26.58%
14.23%
0
10
0
10
10
1
10
2
10
3
10
4
10
0
10
10
1
10
10
2
10
3
10
4
FL1-H
2
1
10
85.84%
4
D
K04 (10µg/ml)
E
AP N.C 48.018
8.63%
10
7.52%
4
K06 (10µg/ml)
AP K06 48.016
9.57%
19.02%
0.68%
0
10 0
10
10
1
10
2
10
3
10
4
10
3
10
3
10
10
FL2-H
FL2-H
FL1-H
2
10
1
10
68.86%
2
1
14.99%
32.54%
0
10
0
10
100
90
80
70
60
8.91
12.84
1
10
2
8.58
8.63
7.23
7.52
14.23
14.99
10
3
10
4
10
0
10
1
10
2
10
3
10
4
FL1-H
9.57
19.02
other
38.87
40
20
10
26.58
50
30
38.87%
0
10
FL1-H
Percentage
FL2-H
FL1-H
10
69.96
late apoptosis
early apotosis
68.86
survival
51.67
32.54
10
0
K02
K03
K04
K06
Supplemental figure3. Betulin and its derivatives caused apoptosis in HBL-100 breast
cancer cells.After treating HBL-100 with 10 µg/mL of K02 (B), K03 (C), K04 (D)
and K06 (E) for 48 hours, the cells were double stained with Annexin V and PI for
flow cytometry. The results were quantified for each quadrant. Compared to control
(untreated HBL-100) (A), cells in quadrant I and VI increased in cells treated with
K02, K03, K04 and K06, and the increase was the most significant in cells treated
with K02 (B) and K06 (E). After quantifying all four quadrants, cell number in
quadrant I and VI from K02 and K06 treatment groups increased significantly
compared to that from K03 and K04 treatment groups and control group (F).
Supplemental figure 4
D
D
PUMA
PERP
PUMA
36hr
0hr
24hr
12hr
36hr
0hr
NOXA
24hr
12hr
36hr
0hr
BAX
24hr
12hr
36hr
0hr
24hr
12hr
36hr
0hr
24hr
12hr
ΔNp63
PERP
TAp63
ΔNp63
BAX
NOXA
PUMA
10
PERP
TAp63
ΔNp63
BAX
NOXA
PUMA
36hr
24hr
0hr
12hr
36hr
24hr
0hr
12hr
36hr
24hr
12hr
0hr
36hr
24hr
12hr
0hr
36hr
24hr
0hr
12hr
1
36hr
36hr
24hr
0hr
12hr
36hr
24hr
12hr
0hr
36hr
24hr
0hr
12hr
36hr
24hr
12hr
0hr
36hr
24hr
12hr
0hr
36hr
0hr
24hr
12hr
1
K04
24hr
10
100
0hr
C
K03
12hr
100
Relative mRNA expression (normalized to GADPH)
B
Relative mRNA expression (normalized to GADPH)
36hr
TAp63
0.1
0.1
0.1
1
0hr
36hr
0hr
24hr
12hr
36hr
0hr
NOXA
24hr
12hr
36hr
0hr
BAX
24hr
12hr
36hr
24hr
0hr
ΔNp63
12hr
36hr
24hr
0hr
TAp63
12hr
36hr
0hr
24hr
1
10
24hr
10
K06
100
12hr
K02
12hr
Relative mRNA expression (normalized to GADPH)
100
Relative mRNA expression (normalized to GADPH)
A
PERP
0.1
Supplemental figure 4. Betulin and its derivatives affected apoptotic-related gene
expression in MDA-MB-231 breast cancer cell line on RNA levels
After treating MDA-MB-231 with 10 µg/mL of K02 (A), K03 (B), K04 (C) and
K06(D) for 0, 12, 24 and 36 hours, real-time PCR was performed for the analysis on
bax, puma, noxa, perp, TAp63 and ΔNp63 gene expression. In MDA-MB-231, bax,
puma, noxa, and perp gene expression increased as K02 (A) and K06 (B) treatment
time increased; whereas the expression of TAp63 and ΔNp63 decreased as the K02 (A)
and K06 (B) treatment time increased. The expression of bax, puma, noxa, perp and
ΔNp63 did not change in K03 (B) and K04 (C) treated cells, but the expression of
TAp63 decreased along with the treatment time.
Supplemental figure 5
100
TAp63
ΔNp63
BAX
NOXA
PUMA
ΔNp63
BAX
PUMA
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
NOXA
PERP
0.1
100
100
K03
B
10
K04
C
10
TAp63
ΔNp63
BAX
NOXA
PUMA
PERP
TAp63
ΔNp63
BAX
NOXA
PUMA
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
1
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
1
0hr
12hr
24hr
36hr
Relative mRNA expression (normalized to GADPH)
TAp63
PERP
0.1
0.1
1
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
0hr
12hr
24hr
36hr
1
10
0hr
12hr
24hr
36hr
10
K06
D
0hr
12hr
24hr
36hr
A
Relative mRNA expression (normalized to GADPH)
K02
Relative mRNA expression (normalized to GADPH)
Relative mRNA expression (normalized to GADPH)
100
PERP
0.1
Supplemental figure 5. The effect of Betulin and its derivatives on apoptotic-related
gene expression in human normal mammary epithelial cell line H184B5F5/M10
After treating H184B5F5/M10 with 10 µg/mL of K02 (A), K03 (B), K04 (C) and
K06(D) for 0, 12, 24 and 36 hours, real-time PCR was performed for the analysis on
bax, puma, noxa, perp, TAp63 and ΔNp63 gene expression. In H184B5F5/M10, the
expression of bax, puma, noxa and perp showed no change with K02(A) and K06(B)
treatment; whereas TAp63 and ΔNp63 expression decreased along with the K02(A)
treatment time. In K06 (B) treatment group, TAp63 expression decreased and the
expression ofΔNp63 increased first and decreased afterwards. In K03 (B) and K04 (C)
treated cells, bax, puma, noxa, perp and ΔNp63 expression was not affected by the
treatment time, and the expression of TAp63 decreased along the treatment time.
Supplemental Table 1 Primers and antibodies list
Primer list of real time PCR
Forward primer
Reverse primer
TAp63
CAGTCCAGAGGTTTTCCAGCAT
TCAATGGGCTGAACATATAG
Bax
ATGTTTTCTGACGGCAACTTC
ATCAGTTCCGGCACCTTG
Puma
ACCTCAACGCACAGTACGA
GAGATTGTACAGGACCCTCCA
Noxa
GGAGATGCCTGGGAAGAAG
CCTGAGTTGAGTAGCACACTCG
Perp
TGTCTTCCTGAGAGTGATTGGA
ACCAGGGAGATGATCTGGAA
GAPDH
CCACTCCTCCACCTTTGAC
ACCCTGTTGCTGTAGCCA
Supplemental Table 2