SUPPLEMENTARY INFORMATION
1. Table S1. Sequences of primers
2. Supplementary figure legend
Figure S1, related to Figure 1
Figure S2, a: related to Figure 1, b: related to Figure 2
Figure S3, related to Figure 2
Figure S4, related to Figure 3, 4
Figure S5, related to Figure 6
Figure S6, related to Figure 6
Figure S7, schematic of the model
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Table S1. Sequences of primers used for quantitative Real-time PCR
p38α
Forward
Reverse
p38β
Forward
Reverse
p38γ
Forward
Reverse
p38δ
Forward
Reverse
MKK3 Forward
Reverse
MKK6 Forward
Reverse
β-parvin Forward
Reverse
α-PIX
Forward
Reverse
L32
Forward
Reverse
GGT TAC GTG TGG CAG TGA AG
AAC GTC CAA CAG ACC AAT CA
GCC ATA TGA TGA GAG CGT TG
GTG AGC TCC TTC CAC TCC TC
AGA TGA TCA CAG GCA AGA CG
TGT AGT TCT TGG CCT CAT CG
TCC TCA GCT GGA TGC ACT AC
TGG TCC AGG TAA TCT TTC CC
GTC GAA GCC ACC CGC AC
CCT CAA AGT TTC TGT CTC CAA TGG
GAA CTG GGA CGA GGT GCG TA
TTT ACT GTG GCT CGG ATC CG
AGA CAC CCA GCT TGA GGA GA
CTG CCA GTT TTT CCA AGA GC
AGG CAT GGG AAG GAG AAG AT
GTC ACC ACC GTT CCT GCT TAT
TAC TGT GCC GAG ATG CTC G
AAA ACG TGC ACA TGA GCT GC
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Figure S1. Expression of p38 MAPK isoforms, MKK3, MKK6, β-parvin and α-PIX in a panel of
normal urothelial, papillary and transitional cancer cell lines. Total RNA from cell lines were
analyzed for mRNA expression of (a) p38 isoforms p38α, p38β, p38γ and p38δ; (b) p38 upstream
activator kinases, MKK3 and MKK6; and (c) β-parvin and α-PIX, by quantitative real-time RTPCR. Human ribosomal housekeeping gene L32 mRNA expression was used as an internal control
to normalize all determined mRNA levels. Cell lines were derived from the indicated origins: SVHUC1 is normal urothelium immortalized with SV-40, RT4 is derived from a bladder papilloma;
5637 is derived from a non-invasive superficial bladder cancer; HT1376 from grade II transitional
bladder cancer; J82 from an anaplastic tumor; TCCSUP is from grade IV metastatic tumor;
UMUC3 is from invasive transitional cancer; TSU-Pr1 is from invasive bladder cancer; U87: grade
IV human glioblastoma. (d) The positive controls for each p38 isoform antibody were determined
by western blotting lysates from selected cell lines (TSU-Pr1; SW480 Duke’s B human colorectal
cancer; PANC-1 human pancreatic cancer; MEF mouse embryonic fibroblast; U87 glioblastoma).
(e) The protein levels of MKKs were measured by western blotting lysates from cell lines indicated.
For qPCR results, n = 3, Error bars are S.E.M. For western blot, n=3.
Figure S2. Bright-field pictures of the TSU-Pr1 series, ILK and p38 siRNA did not affect TSUPr1 cell viability. (a) Bright-field photographs of the TSU-Pr1 series at ×10 magnification. TSUPr1 cells exhibit fibroblastoid morphology, whereas B1 and B2 cells exhibit a cuboidal epithelial
morphology (Chaffer, Brennan et al. 2006). (b) TSU-Pr1 cells were transfected with NTC or
targeted siRNA as indicated for 48 h, and cell viability was assessed using an ATP assay as
described in Materials and Methods. n=3, Columns: mean; Error bars: S.E.M.
Figure S3. Ectopic p38 expression partially improved directional migration in ILK deficient cells.
TSU-Pr1 cells were transfected with non-target control (NTC) or ILK siRNA for 48 h followed by
transfection with FLAG-p38 or pcDNA control vector for 24 h. Then cells were added into the
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upper wells of microchemotaxis chambers. Bottom chambers contained medium supplemented with
10% FBS. (a) Representative images of crystal violet stained cells that have migrated toward 10%
FBS. (b) Cell counts were obtained using Image J software. n=3, Columns: mean; Error bars:
S.E.M. *P =0.002, #P=0.005. (c) Western blot confirmed depletion of ILK protein and ectopic
expression of p38. The levels of p38 protein expression were calculated relative to GAPDH
using densitometry. n=3.
Figure S4. No difference in serum induced Rac1 activation was observed in cells treated with
siRNAs. (a) TSU-Pr1 cells were starved overnight then pretreated for 2 h with the indicated
concentrations of the Rac1 inhibitor NSC23766. The cells were then assayed for serum-induced
microchemotactic migration, and numbers of migrated cells were counted using Image J. n=3,
Columns: mean; Error bars: S.E.M. *P=0.018, **P <0.001. (b) Total cell lysate of TSU-Pr1 was
loaded with non-hydrolysable GTP analog (GTPγS) or GDP as a positive or negative control for the
pull-down assay as described in Materials and Methods. (c) After starvation as described in
Materials and Methods, TSU-Pr1 cells were then treated with EGF (10ng/ml as final concentration)
for times indicated. Whole-cell lysates were assayed for Rac1 activation. (d) TSU- Pr1 cells were
transfected with NTC, ILK, p38 or p38 siRNA, starved as described in Materials and Methods.
Whole-cell lysates were assayed for Rac1, CDC42 and RhoA GTPase activation as described in
Materials and Methods. ILK and p38β expression were analyzed by western blotting with a 10 µg
sample of each whole-cell lysate. The level of expression of each activated Rho GTPase protein
was calculated relative to GAPDH using densitometry and values shown between each blot. n=3.
Figure S5. Subcellular distributions of p38 isoforms in bladder cancer cells. (a) FLAG-tagged p38α,
p38β, p38γ and p38δ of each expression plasmid was transfected into TSU-Pr1 cells. 24 h posttransfection, the cells were fixed and stained with the mouse anti-FLAG M2 antibody and goat antimouse secondary antibody (green). Nuclei were identified by DAPI staining (blue). Scale bar, 20
µm. (b) Western blot analysis of TSU-Pr1-B1 cells after subcellular fractionation. Blots show
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representative results, indicating endogenous ILK in both fractions and p38β in the cytoplasmic
fraction. α-Tubulin and HDAC1 were used as markers for cytoplasmic and nuclear compartments
respectively.
Figure S6. Full-length blots for co-immunoprecipitation in Figure 6a are shown.
Figure S7. Model for regulation of bladder cancer cell migration by ILK-p38β signaling complexes.
ILK sequesters and stabilizes p38β in a cytoplasmic complex that may act downstream of Rac1.
Serum stimulates ILK/p38 signaling and activation of Hsp27, which is dampened in ILK-inhibited
cells. The ILK inhibitor QLT-0267 prevents serum-stimulated ILK-p38 complex formation and
inhibits cell chemotactic migration.
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