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Supplementary figure legends
Figure S1.A. Effect of pimasertib followed by short treatment with gemcitabine in vitro.
The MTT assay was used to assess the effect of 4-hour pre-treatment with 500nM
pimasertib followed by 24h exposure with increasing concentrations of gemcitabine (5-2550nM), on the growth of BxPC-3 cells. Combination Indices (CI) describing drug
combinations were calculated from the fraction affected of three independent experiments.
Figure S1.B. Apoptotic effect of simultaneous administration of pimasertib with
gemcitabine.
Caspase 3/7 activity was used to detect apoptosis in BxPC-3 cells after 48h simultaneous
treatment of 500nM pimasertib with 50nM gemcitabine. Results are presented as fold
increase to untreated sample and are shown as mean  SD (n=3) (P≥ 0.05 ns).
Figure S2.A. MEK inhibitor AS703988 induces downregulation of RRM1 protein
Immunoblotting analysis of RRM1 protein expression after 24h treatment with 1µM of
MEK1/2 inhibitor AS703988 (EMD Serono), 50nM gemcitabine alone or in combination with
1µM AS703988 in BxPC-3 cells. Immunoblotting analysis was performed on whole-cell
lysates. Calnexin was used as loading control. Results are representative of three
independent experiments.
Figure S2.B. RRM1 expression is not modulated by EGFR signalling.
BxPC-3 cells were treated with the EGFR inhibitor gefitinib (1µM). 24 hours later cells were
harvested and protein lysates were analyzed by immunoblotting with the indicated
antibodies and calnexin was used as loading control.
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Figure S2.C. Pimasertib effect on RRM1 protein expression in human pancreatic cancer cell
lines.
MIAPaCa-2 and SUIT-2 cells were treated with 1µM pimasertib and extracted at different
time points (30min to 4h). Total lysates were analysed by immunoblotting with the
indicated antibodies and calnexin was used as loading control.
Figure S3.A. MG132 impairs RRM1 downregulation induced by pimartib in PANC-1 cells.
PANC-1 cells were pre-treated with 1µM of proteasome inhibitor MG132 for 1 hour
followed by a 4-hour treatment with 1µM pimasertib or pre-treated for 1 hour with 100
µg/ml cycloheximide followed by 24 hour treatment with 1µM pimasertib. Protein lysates
were analysed by immunoblotting analysis with the indicated antibodies and calnexin was
used as loading control. Results are representative of two independent experiments.
Figure S4.A. Knockdown of P53 with siRNA does not affect pimasertib-mediated RRM1
downregulation.
BxPC-3 cells were transfected with specific siRNA targeting P53 (50nM) or scrambled. 72
hours after transfection cells were treated for 24h with 1µM pimasertib. Protein lysates
were analysed by immunoblotting with an anti-P-53 antibody and calnexin was used as
loading control.
Figure S4.B. Reduction of RRM1 protein induced by MEK inhibition is mediated by AKT.
SUIT-2 cells were treated for 4 hours with 1µM of PI3K inhibitor GDC-0941 together with
1µM pimasertib and whole cell lysates were immunoblotted using the indicated antibodies
and β-actin was used as loading control.
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Figure S5.A. Inhibition of tumor growth in mice treated with 15mg/kg pimasertib plus
80mg/kg gemcitabine.
TB32048 tumor-bearing mice were treated for 12 days with vehicle, 15mg/kg pimasertib
(daily) by oral gavage, gemcitabine 80mg/kg (twice/week) intraperitonally or a sequential
combination of pimasertib 15mg/kg followed by gemcitabine 80mg/kg. After sacrifice,
tumor weight was measured. Student’s t test was used to determine the statistical
significance of differences in tumor weight (gemcitabine vs gemcitabine/pimasertib
**P<0.01).
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