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Supplementary Methods
Bromo-desoxyuridine (BrdU) incorporation immunohistochemistry. Primary
BMSCs from proliferating cultures were freshly seeded at low density and left to grow
until clear signs of continued proliferation were visible. Cells were then treated with
either 20 µM QLT0267 or with DMSO for 2 d, after which time in some of the cultures
the drug was removed and exchanged for drug-free full medium. BrdU (final
concentration 10 µM) was added twice (at days 3 and 4) and the BMSCs were
cultured for a total of 5 days. Cells were washed with PBS, fixed in 70% EtOH / 2N
HCl for 15 min at room temperature, again washed with PBS, treated for 5 min with
1% H2O2 in PBS to deactivate potential endogenous peroxidases, washed 3 x with
PBS/10% FBS, and incubated with peroxidase-coupled anti-BrdU antibody (Roche,
Mannheim, Germany; no. 11585860001) for 1 h at room temperature. After 3 washes
with PBS the signal was developed by incubation with 3,3-diaminobenzidine solution
(Kem-En-Tec Diagnostics, Taastrup, Denmark). For microphotography, cells were
washed 3 times with PBS, lightly counterstained with crystal violet solution, again
washed with PBS, covered with glycerol and immediately photographed (Nikon
Eclipse TS100 microscope and Nikon NIS Elements software, version 2.31). The
whole procedure was performed in the original culture wells (24 well plates).
Construction of ILK expression vectors. The human ILK gene was amplified with
Pfu DNA polymerase using first strand cDNA derived from MM cell line U266 as
template.
The
primers
5’-GGACGATATCATGGACGACATTTTCACTC-3’
(ILK
forward) or 5’-ACGCGTATACGGACGACATTTTCACTC-3’ (HA-ILK forward) were
used
in
combination
5’-GACCGCGGCCGCCTACTTGTCCTGCATCTTC-3’
with
(reverse)
primer
to
amplify
sequences suitable for construction of either wild-type or of 5’-3xHA-tagged ILK
expression vectors (28 cycles of denaturation at 94°C (30 seconds), annealing at
62°C (1 min) and extension at 72°C (3 min)). Purified PCR products were 3’-A-tailed
in a reaction with Taq DNA polymerase and 200 nM dATP (72°C for 30 min), cloned
into pGEMTeasy (Promega, Mannheim, Germany), and suitable clones identified by
restriction digests and subsequent verification of the complete coding sequence for
ILK. The pCAGGS/SE expression vector (Stühmer et al. (2002), Development,
129:245-252) for wildtype ILK was then generated by excising the respective insert
from pGEMTeasy with EcoRV/NotI (sites introduced with the PCR primers, see
1
underlined sequences above) and subcloning into EcoRV/NotI-cut pCAGGS/SE. To
construct the 3x-HA-tagged ILK variant, the respective sequence was excised from
pGEMTeasy with Bst1107I and NotI (sites introduced with the PCR primers, see
underlined sequences above) and subcloned into a pCAGGS-3xHA-expression
vector [20]. Of note, the Bst1107I site used for cloning is not in frame, and the
original ILK start codon was omitted.
ILK overexpression in INA-6 cells and treatment wih QLT0267. INA-6 cells were
transiently transfected with expression vectors pCAGGS-ILK (10 µg/ml) or pCAGGSHA-ILK (10 µg/ml) using the standard electroporation procedure (see Methods
section). Cells transfected with empty pCAGGS/SE vector served as controls. Cells
were co-transfected with an expression plasmid for truncated CD4 for purification of
strongly transfected cells through MACS microbead selection [26].
2