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Supplementary Figures
Supplementary Figure S1. The chemical structure of tanshinone-1.
Supplementary Figure S2. Tanshinone-1-mediated cellular apoptosis was independent
of drug transporters. (a) Cells were treated with 20 μM tanshinone-1 for the indicated time.
The KB and KB/VCR cells were then stained with Annexin V/PI and analyzed by flow
cytometry; mean ± SD; *, p < 0.05; **, p < 0.01. (b) Tanshinone-1 accumulated in KB and
KB/VCR cells. Cells were exposed to 80 μM tanshinone-1 for the indicated time, and
analyzed by high-performance liquid chromatography. The results were presented as mean ±
SD for three independent experiments. (c) Tanshinone-1 did not change the gene expression
of drug transporters. KB/VCR cells were treated with gradient concentrations of tanshinone-1
for 12 h. Cellular RNA extracted from the cells were subjected to reverse transcription
polymerase chain reaction (RT-PCR). The resulting cDNA were analyzed by gel
electrophoresis.
Supplementary Figure S3. MDR cells transfected with Stat3 siRNA were resistant to
vincristine and adriamycin and constitutively-activated Stat3 did not significantly affect
the sensitivity of parental KB cells to tanshinone-1. (a, b) MCF7/ADR and KB/VCR cells
were transfected with siStat3 for 24 h and then treated with adriamycin (ADR) (a) or
vincristine (VCR) (b) for another 48 h. SRB assays were done and the IC50 was expressed as
mean ± SD. (c-e) Constitutively activated mouse Stat3 (M3C) did not change the sensitivity
of parental KB cells to tanshinone-1. Cells were transfected with M3C for 24 h. Then the cells
were treated with 20 μM tanshinone-1 for 48 h and analyzed by SRB assays. The IC50 was
expressed as mean ± SD (c). The cells were transfected with M3C for 48 h and then treated
with 20 μM tanshinone-1 for 24 h. Flow cytometry was done for apoptosis analyses, and the
apoptosis rates from three independent experiments were expressed as mean ± SD (d). The
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cells were transfected with M3C for 48 h. Then the cells were treated with 20 μM tanshinone1 for 4 h followed by Western blotting (e); the ectopic Stat3 (EC-Stat3) expressed by M3C
contained green fluorescent protein and thus possessed bigger molecular weight than the
endogenous Stat3 (EN-Stat3). (f-h) Constitutively-activated human Stat3 (H3C) did not
change the sensitivity of KB cells to tanshinone-1. Cells were transfected with H3C or empty
vector pCMV for 24 h, and then treated with 20 μM tanshinone-1 for 48 h followed by SRB
assays. The IC50 was expressed as mean ± SD (f). Cells were transfected with H3C and
pCMV for 48 h followed by the treatment with 20 μM tanshinone-1 for another 24 h, stained
with Annexin-V/PI, and then analyzed by flow cytometry (g). The apoptosis rates were
expressed as mean ± SD (g, upper panel); representative histograms were shown (g, lower
panel). The cells were transfected with H3C (the molecular weight similar to the endogenous
Stat3) for 48 h, and then, they were treated with 20 μM tanshinone-1 for 4 h followed by
Western blotting (h).
Supplementary Figure S4. The cellular levels of Shp1, Shp2, and PTP1B phosphatases
in three pairs of MDR and corresponding parental tumor cells. Hep-3B cells were used
for reference.
Supplementary Figure S5. The impacts of p38, AKT, and ERK inhibitors on
tanshinone-1-mediated enhancement of cellular p38, AKT, and ERK phosphorylation.
KB cells were pretreated with PI103 (10 μM, AKT) or AZD6244 (5 μM, ERK) for 30 min or
SB203580 (50 μM, p38) for 10 min. The cells were then exposed to 20 μM of tanshinone-1
for 15 min (p-p38 and p-ERK) or 4 h (p-AKT and p-705-Stat3) and analyzed by Western
blotting.
Supplementary Figure S6. Schematic representations of the anticancer and anti-MDR
effects of tanshinone-1 in combination with p38, AKT, and ERK inhibitors and the
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proposed strategy for overcoming MDR. Solid lines, direct experimental evidence; broken
lines, inferential relationships. (a) Tanshinone-1 activates Shp2 and PTP1B via unknown
mechanism(s), therefore reducing p-705-Stat3 which elicits anticancer and secondarily
activates p38-, AKT-, and ERK-involved signaling networks. A combination of p38, AKT,
and ERK inhibitors (especially p38 and AKT inhibitors) could eliminate the compensatory
activation and potentiate the effects of tanshinone-1 on MDR tumor cells. This feature could
be useful for multitarget drug design. (b) Comparison of primary characteristics of the
classical strategy and the proposed strategy using anti-MDR drugs for MDR circumvention.
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