Download Supplementary Figure Legends Figure 1. Inhibition of FGF

Supplementary Figure Legends
Figure 1.
Inhibition of FGF-dependent signalling and proliferation in
fibroblasts by AZ6089 and AZ8010.
(A) CCL39 cells were serum starved for
24hrs, then left in serum free medium or re-stimulated with 10ngml-1 FGF2, either
alone or in the presence of AZ6089, AZ8010, PD173074 (PD) or AG1478 (AG), for a
further 24hrs.
(B) Serum-starved CCL39 cells were treated with the indicated
concentrations of AZ6089 or AZ8010 before stimulation with 10ngml-1 FGF2 for a
further 24hrs.
For (A & B) 5μM thymidine containing 0.5μCi [3H]thymidine was
added for the last 6hrs of treatment. [3H] thymidine incorporation was quantified by
liquid scintillation counting and data points represent mean ± standard deviation of
triplicate determinations.
(C) Serum-starved CCL39 cells were treated with the
indicated concentration of AZ6089 or AZ8010, 100nM PD173074 (PD) or 100nM
AG1478 (AG) for 30 mins and then stimulated with 10ngml-1 FGF2 for 1hr. (D)
Serum-starved CCL39 cells were treated with 100nM AZ6089, 100nM AZ18010,
100nM PD173074 (PD) or 1μM Selumetinib/AZD6244 (Sel) for 30 mins prior to
stimulation with 10ngml-1 FGF2 for the indicated times. For (C & D) cell lysates were
then fractionated by SDS PAGE and subjected to Western blot analysis with the
indicated antibodies. In all cases results are from a single representative experiment
that was carried out n=3 times with similar results.
Figure 2. AZ6089 and AZ8010 selectively inhibit FGFR-driven proliferation in
fibroblasts.
(A) Serum-starved CCL39 cells were treated with 100nM AZ6089,
100nM AZ18010, 100nM PD173074 (PD) or 1μM Selumetinib/AZD6244 (Sel) or
100nM (AG1478) for 30 mins prior to stimulation with 10ngml-1 FGF2, 100nM
thrombin, 100nM EGF or 10ngml-1 PDGF for 24 hours. 5μM thymidine containing
0.5μCi [3H]thymidine was added for the last 6hrs and [3H] thymidine incorporation
was quantified by liquid scintillation counting. Data points represent mean ± standard
deviation of triplicate determinations. (B-D) Serum-starved CCL39 cells were treated
with 100nM AZ6089,
100nM AZ18010,
100nM PD173074 (PD)
or
1μM
Selumetinib/AZD6244 (Sel) or 100nM (AG1478) for 30 mins as indicated prior to
stimulation with (B) 100nM thrombin, (C) 100nM EGF or (D) 10ngml-1 PDGF for 1
hour. Cell lysates were then fractionated by SDS PAGE and subjected to Western
blot analysis with the indicated antibodies. In all cases results are from a single
representative experiment that was carried out n=3 times with similar results.
Figure 3. Identification of FGFR-dependent multiple myeloma cell lines using
PD173074. (A) The indicated MM cell lines were treated for 24hrs with DMSO
vehicle control or 100nM PD173074 (PD173) in complete medium containing 10%
FBS. 5μM thymidine containing 0.5μCi [3H]thymidine was added for the final 6hrs of
treatment and [3H] thymidine incorporation was quantified by liquid scintillation
counting. Data points represent mean ± s.d. of triplicate determinations. Results are
from a single representative experiment that was carried out n=3 times. (B) Multiple
myeloma cell lines were treated with DMSO vehicle control or 100nM PD173074
(PD173), in medium containing 10% FBS, for 4 days. Cells were incubated with
Annexin-V-FITC and propidium iodide and Annexin-V binding was monitored by flow
cytometry. Data points represent mean ± s.d. of triplicate determinations. Results are
from a single representative experiment that was carried out n=3.
Figure 4. Identification of FGFR-dependent urothelial cancer cell lines using
PD173074 and demonstration of G1 cell cycle arrest elicited by PD173074. (A)
Urothelial carcinoma cell lines were treated for 24hrs with PD173074 at the
concentrations indicated in complete medium containing 10% FBS. 5μM thymidine
containing 0.5μCi [3H]thymidine was added for the final 6hrs of treatment and [3H]
thymidine incorporation was quantified by liquid scintillation counting. Data points
represent mean ± coefficient of variance of duplicate determinations. Results are
from a single representative experiment that was carried out n=3. (B) 97-7 or RT4
cells were treated with DMSO vehicle control or 100nM PD173074 (PD) for 72 hours
in complete medium containing 10% FBS. Cells were then stained with propidium
iodide and their cell cycle distribution assessed by flow cytometry. Data points
represent mean ± range of duplicate determinations and are taken from a single
representative experiment that was carried out n=3. (C) Representative histograms
of 97-7 cells treated for 72hrs with either DMSO control or 100nM PD173074.
Figure 5. Sum52-PE breast cancer cells undergo a G1 cell cycle arrest in
response to PD173074. (A) Sum52-PE, SKBR3 and T47D breast cancer cell lines
were treated with DMSO vehicle or 100nM PD173074 for 24 hours in complete
medium containing 10% FBS. 5μM thymidine containing 0.5μCi [3H]thymidine was
added for the final 6hrs of treatment and [3H] thymidine incorporation was quantified
by liquid scintillation counting. Data points represent mean ± s.d. of triplicate
determinations. Data are from a single representative experiment that was carried
out n=2 times with identical results. (B) Sum52-PE, T47D and SKBR3 cells were
treated with DMSO vehicle control or 100nM PD173074 (PD) for 72 hours in
complete medium containing 10% FBS. Cells were then stained with propidium
iodide and their cell cycle distribution assessed by flow cytometry. Data points
represent mean ± range of duplicate determinations and are taken from a single
representative experiment that was carried out n=3.
Figure 6. Characterisation of KMS-11R cells. (A) KMS-11 and KMS-11R cells
were seeded at 1x105 cells ml-1 in their normal growth media (no drug for KMS-11,
3µM AZ8010 for KMS-11R) and cell numbers were assessed at 24hr intervals. Data
points represent mean ± s.d. of quadruplicate determinations. (B) KMS-11 or KMS11R cells were treated for 24hrs with cisplatin (CisPt), etoposide (Etop) or paclitaxel
(Pac) at the concentrations indicated and in medium containing 10% FBS. (C) KMS11 or KMS-11R cells were treated for 24hrs with PD173074 at the concentrations
indicated and in medium containing 10% FBS. For (B-C) 5μM thymidine containing
0.5μCi [3H-methyl] thymidine was added for the final 6hrs of treatment and [3H]
thymidine incorporation was quantified by liquid scintillation counting. Data points
represent mean ± s.d. of triplicate determinations and results are from a singe
representative experiment that was carried out n=3 times.