Supplementary Figure S1. Sequence analysis of CIB1a clone. cDNA sequence alignment
of CIB1a (marked in black) with CIB1 (marked in green). Sequence analysis and alignment
were done by MacMolly®Tetra, Version 3.9, 1999 (Soft Gene GmbH).
Supplementary Figure S2. (A) Phospho-Serine118-specific antibody characterization by
immunoblotting. Recombinant GST-CIB1a-WT was incubated with GFT-PKD2-WT or
GFP-PKD2-DA or GFP-PKD2-3SE bound on Protein A Sepharose in kinase assay buffer
with cold ATP. Phosphorylation of the recombinant GST-CIB1a was assessed by
immunoblotting with anti-phospho-Serine 118 (p-CIB1a). GFP-PKD2 mutants were isolated
from HEK293-T cells incubated with or without PMA (400nM). Equal levels of proteins
used in reactions were assessed by immunoblotting against anti-EGFP (for GFP-tagged
PKD2), anti-CIB (for GST-CIB1a-WT). (B) Endogenous CIB1a is getting phosphorylated
in response to PMA in a PKD2-dependent manner. PKD2 was silenced in Panc1 cells by
shRNA
directed
against
PKD2
NM_016457.x-294s1c1(sh13D5);
(Sigma-Aldrich,
NM_016457.x-1720s1c1(sh13D4);
NM_016457.x-1767s1c1(sh13D6);
NM_016457.x-
1335s1c1 (sh13D7)). Kinase knock-down was verified by Western blotting with anti-PKD2.
CIB1a was immunoprecipitated from Panc1 cells (1mg of cell lysate) infected with control
shRNA and shPKD2, stimulated with or without PMA (400nm), and IP was verified with
anti-CIB (CIB1a). Phosphorylation of CIB1a in response to PMA was detected by
immunoblotting with anti-phosphoSer118 (p-CIB1a). Graph represent quantification of
integrated density done by ImageJ. Results shown represent the means ± S.E.M. of three
independent experiments (* p<0.05; **p<0.001;***p<0.0001).
Supplementary Figure S3. (A) Phosphorylation of CIB1a at Serine 118 decreases its
binding to PKD2. GFP (lane 1), GFP-CIB1a-WT (lane 2), GFP-CIB1a-S118A (lane 3) or
GFP-CIB1a-S118E (lane 4) were co-expressed with Flag (lane 1) or Flag-PKD2-WT (lane 2, 3
1
and 4) in HEK293-T cells. The proteins were immunoprecipitated with anti-GFP (top left) or
anti-Flag (bottom left) followed by anti-Flag or anti-CIB Western blotting, respectively. To
verify that each tagged protein was expressed WCL were tested with anti-CIB (for GFP
tagged CIB1a, top right) and anti-Flag, respectively (for Flag tagged PKD2, bottom right).
Graphs represent quantification of integrated density done by ImageJ. Results shown
represent
the
means
±
S.E.M.
of
three
independent
experiments
(*
p<0.05;
**p<0.001;***p<0.0001).
Supplementary Figure S4. Expression of phosphomimetic CIB1a mutant partially
rescues tumour growth impaired by PKD2 knockdown. (A) HeLa cells were transduced
with lenti-viruses expressing scramble control shRNA (Sigma-Aldrich scramble, shc002
(shCon))
and
shRNA_PKD2
NM_016457.x-294s1c1(sh13D5);
(Sigma-Aldrich,
NM_016457.x-1720s1c1(sh13D4);
NM_016457.x-1767s1c1(sh13D6);
NM_016457.x-
1335s1c1 (sh13D7)). A PKD2 knockdown was probed using a specific anti-PKD2 antibody
following selection. Blots were re-probed for Actin used as loading control. (B) Images of
tumours developed on CAM. Semi-stable scramble control shRNA (shCon) and shPKD2
expressing HeLa cells were transfected with CIB1a-WT-IRES-GFP, CIB1a-S118A-IRES-GFP
or CIB1a-S118E-IRES-GFP and applied in silicon rings on the CAM. Representative images
of tumours are shown. (C) Graph represents quantification of the tumour areas within silicon
rings. The dashed line indicates the quantified tumour area. Results shown represent the
means ± S.E.M. of at least three tumours from three independent experiments (* p<0.05;
**p<0.001;***p<0.0001). (D) Aliquots of cells used in the CAM assay were lysed and tested
for equal expression of the CIB1a mutants and PKD2 knockdown. Membranes were reprobed
with a β-actin antibody. (E) CIB1a does not significantly stimulate DNA synthesis of Hela
cells. Hela cells were transfected with EGFP-vector, IRES-CIB1a-WT-GFP, IRES-CIB1aS118A-GFP or IRES-CIB1a-S118E-GFP plasmids and cultivated under normoxic or hypoxic
2
conditions. Subsequently the GFP-positive population was sorted, stained with propidium
iodide and subjected to flow cytometric analysis as described in materials and methods. Bars
are the means +/- SEM of the three independent experiments (T-test: *p<0.05; **p<0.001)
Supplementary Figure S5. CIB1a interacts with FAK, but mutation within CIB1a does
not affect significantly its binding capacity to the kinase. GFP-CIB1a-WT (lane 1), GFPCIB1a-S118A (lane 2), GFP-CIB1a-S118E (lane 3) or GFP (line 4) were co-expressed with
Myc-FAK (lane 1, 2 and 3) or Myc (lane 4) in HEK293-T cells. The proteins were
immunoprecipitated with anti-GFP (top left) or anti-Myc (bottom left) followed by anti-Myc
or anti-CIB Western blotting, respectively. Proteins expression was verified in WCL by
immunoblotting with anti-Myc (for FAK, top right) and anti-GFP, respectively (for CIB1a,
bottom right). Graphs represent quantification of integrated density done by ImageJ. Results
shown represent the means ± S.E.M. of three independent experiments (* p<0.05;
**p<0.001;***p<0.0001). (B) Immunoprecipitate kinase assay using mock- and HEK293-T
cells co-transfected with CIB1a mutants and Myc-FAK. Although FAK expression levels are
comparable (WB: Myc, for Myc-FAK), the amount of phosphotyrosine is up-regulated on
CIB1a overexpression (WB: pY). CIB1a mutations did not further affect FAK activation.
CIB1a protein expression was verified in WCL by immunoblotting with anti-CIB.
Supplementary Figure S6. CIB1a interacts with PAK1, but mutation within CIB1a does
not affect significantly its binding capacity to the kinase. GFP-CIB1a-WT (lane 1), GFPCIB1a-S118A (lane 2), GFP-CIB1a-S118E (lane 3) or GFP (line 4) were co-expressed with
Myc-PAK1 (lane 1, 2 and 3) or Myc (lane 4) in HEK293-T cells. The proteins were
immunoprecipitated with anti-GFP (top left) or anti-Myc (bottom left) followed by anti-Myc
or anti-CIB Western blotting, respectively. Protein expression was verified in WCL by
immunoblotting with anti-Myc (for PAK1, top right) and anti-GFP, respectively (for CIB1a,
3
bottom right). Graphs represent quantification of integrated density done by ImageJ. Results
shown represent the means ± S.E.M. of three independent experiments (* p<0.05;
**p<0,001;***p<0, 0001). (B) CIB1a does not regulate PAK1 kinase activity.
Immunoprecipitate kinase assay using mock- and HEK293-T cells co-transfected with CIB1a
mutants and Myc-PAK1 (upper panel). Myelin basic protein (MPB) was used as a PAK1
substrate. Protein expression was verified in WCL by immunoblotting with anti-CIB (for
CIB1a) and anti-Myc (for PAK1). Graphs represent quantification integrated density done by
AIDA Image Analyzer. Results shown represent the means ± S.E.M. of three independent
experiments (* p<0.05; **p<0.001;***p<0.0001).
Supplementary Figure S7. (A) CIB1a stimulates Nur77 transcription, however, there is
with no further change in Nur77 promoter activity upon CIB1a phosphorylation by
PKD2. HeLa cells were co-transfected with a luciferase reporter plasmid driven by the Nur77
promoter (pNur77-Luc) and CIB1a mutants (WT, S118A or S118E) or PKD2-3SE (positive
control). Luciferase activities were determined and normalized based on Renilla luciferase
expression and represented as relative light units (RLUs) of firefly luciferase. (B) CIB1a
phosphorylation by PKD1 has no effect on hypoxia-induced VEGF-A reporter activity.
HeLa cells co-transfected with pGL2-VEGF-luc, pTK-Renilla and wild type (CIB1a-WT),
non-phosphorylatable (CIB1a-S118A) or phosphomimetic CIB1a mutants (CIB1a-S118E) were
incubated under normoxic or hypoxic conditions for 24 h followed by luciferase activity
assays. (C) Phosphorylation of CIB1a does not affect FGF2 secretion by epithelial
tumour cells. Cancer cells were transfected with wild type (CIB1a-WT), the nonphosphorylatable (CIB1a-S118A), or phosphomimetic CIB1a mutant (CIB1a-S118E),
respectively. Cells were subsequently incubated under normoxic or hypoxic conditions for
12h as indicated. FGF2 was determined in supernatant of the tumour cells using a specific
ELISA. Results are representative for three independent experiments each performed in
4
triplicates. Protein expression was verified in WCL by immunoblotting with anti-CIB (for
CIB1a) and anti-GFP (for PKD2). Bars are the means +/- SEM of the three independent
experiments (T-test: *p<0.05; **p<0.001)
Supplementary Figure S8. (D) Basal level of CIB1a phosphorylation at Ser118 in various
tumour cell lines. CIB1a was immunoprecipitated from equal amount of cell lysate (500µg)
from the respective cell lines, and IP was verified by anti-CIB1a. Baseline phosphorylation
level was tested by immunoblotting with anti-phospho CIB1a-Ser118 antibody.
5