Supplementary material:
Materials and Methods
Antibodies. Anti-STAT1, anti-p-STAT2, anti-PKR, anti-p-Erk1/2, anti-Erk1/2 (Cell Signaling
Technologies; Beverly, MA, USA), anti-p-PKR (Invitrogen; Carlsbad, CA), anti-H-Ras, anti-OAS1,
anti-eIF2Bε (Santa Cruz Biotechnology; Santa Cruz, CA, USA), anti-STAT2 (BD Transduction
laboratories; San Diego, CA, USA), anti-MxA (Proteintech Group Inc.; Chicago, IL, USA),anti-Rac1
(Gene Tex Inc.; San Antonio,TX, USA), anti-NDV polyclonal antisera (gift from Angelika Roemer
Oberdoerfer; FLI, Island Riems, Germany), anti-GAPDH (Chemicon International Temecula; CA,
USA); anti-actin (Sigma-Aldrich; St. Louis, MO, USA), anti-rabbit IgG, anti-mouse IgG and anti-goat
IgG secondary antibodies labeled with Alexa Fluor 680 (Invitrogen), anti-mouse IgG Cy2, anti-rabbit
IgG Cy3 (Jackson-ImmunoResearch; West Grove, PA, USA).
Plasmids. The sequence confirmed Rac1 cDNA clone AB3094-A01 (OriGene Technologies Inc.;
Rockville, MD, USA) provided in a pCMV6-XL5 vector was cloned into the pcDNA3.1(-)hygromycin B expression vector (Invitrogen) using the Not I restriction sites. The dominant negative
Rac1T17N mutant (Ridley et al., 1992) was generated by using the QuikChange II XL Site-directed
Mutagenesis Kit (Stratagene; La Jolla, CA, USA) according to the manufacturer’s instructions.
Cell lines and culture conditions. Immortalized human keratinocytes (HaCaT) and derivative cells
(DKFZ) as well as MDA-MB-231 cells (ATCC) were maintained in DMEM/ HAM’s F12 culture
medium with stable glutamine (Biochrom AG; Berlin, Germany) and 10 % FCS. Stable selection of
transduced HaCaT cells was performed by using 0.5 µg/ ml puromycin and selection of nucleofected
HaCaT and RT3 K1 cells by using 300 µg/ ml hygromycin B. Clonal selection of HaCaT H-RasV12
cells was performed by serial dilutions. HT1080 and HCT 116 cells (ATCC) were cultured in DMEM
with stable glutamine (Biochrom AG) and 10 % FCS.
Electroporation of cells. Rac1wt or Rac1T17N (over)expressing HaCaT and RT3 K1 cells were
generated by using AMAXA`s nucleofection technology (Cologne, Germany) and AMAXA’s
optimized nucleofection protocol for HaCaT cells.
Lentiviral transduction of HaCaT cells. Cells seeded in 6-wells were transduced with 1 µg (p24)
pLenti6_puromycin-HrasV12 or pLenti6_puromycin-JRED transduction control (kindly provided by
Ulrike Ulbricht, Bayer Schering Pharma AG) in 500 µl growth medium in the presence of polybrene
(Sigma-Aldrich) with a final concentration of 6 µg/ ml. 5 h post infection cells were washed and fresh
growth medium was added.
Western Blot analysis. After electrophoretic separation and immunoblotting of whole cell lysates (50
µg) blots were incubated with appropriate primary antibodies and secondary antibodies labeled with
Alexa Fluor 680. Visualization of bound antibodies was performed by using the Odyssey® Infrared
Imaging System (LI-COR Biosciences; Bad Homburg, Germany).
Colony Formation assay in soft agar. 1x104 cells were resuspended in 2 ml of growth medium
containing 0.4 % agarose (Cambrex Bio Science Rockland; Rockland, ME, USA) and 10 % FCS. Cell
suspensions were plated on previously solidified 0.6 % agarose layers in 6-well plates and were
incubated for 4 weeks before colony formations were monitored by inverse optical microscopy using a
2.5x plan neofluar objective.
Xenograft experiment. The tumor growth of HaCaT, RT3 and RT3 K1 cells was determined by
subcutaneous inoculation of 5x106 cells mixed with Matrigel (BD Bioscience; San Jose, CA, USA)
into the right flank of female NMRI nude mice (Taconic; Germantown, NY, USA). The measurement
of tumor area was performed using a caliper. Animal studies were conducted in accordance with the
guiding principles in the care and use of animals htpp://www.theaps.org/about/opguide/appendix.htm#care.
Virus and virus infection experiments. The virus strain NDV MTH-68 was obtained from Laszlo
Csatary (Hungary). Recombinant EGFP expressing MTH-68 was designed as previously described by
(Puhler et al., 2008). Luciferase expressing MTH-68 was designed equally but the EGFP transgene
was replaced by Firefly luciferase amplified from pTA-Luc (Clontec laboratories; Palo Alto, CA,
USA) with the primers Luc fw 5´-ccttaattaaccaccatggaagacgccaaaaacataaagaaagg-3´ and Luc rev 5´ccttaattaagcgcttacacggcgatctttccgccc-3´ containing flanking Pac1 recognition sites. The PCR product
with Pac1 sites was cloned into the pflMTH-68 Pac vector yielding pflMTH126. Virus rescue was
performed as described previously (Romer-Oberdorfer et al., 1999).
Infection of confluent cells was performed as described previously (Puhler et al., 2008). Viral EGFP
expression was detected by fluorescence microscopy and quantification of viral luciferase activity by
performing a Steady-Glo® Luciferase Assay (Promega; Madison, WI, USA) according to the
manufacturer’s instructions.
Determination of virus yield in supernatants. Determination of virus titers was performed as
described previously (Puhler et al., 2008).
Drug treatment and infection. Cells grown in 96-wells were pretreated with small molecule
inhibitors targeting Rac1 [NSC 23766; Calbiochem; San Diego, USA; (Gao et al., 2004)], PI3K
[LY294002; Invitrogen] or MEK 1/2 [U0126; Calbiochem] for 1 h. Afterwards, cells were infected
with NDV-luciferase in the presence or absence of inhibitor.
Immunofluorescence of Rac1wt-overexpressing and infected cells. HaCaT Rac1wt-pool cells
grown in 96-wells were infected with NDV for 24 h. Infected cells were then washed with PBS
(Biochrom AG), fixed with 3.7 % paraformaldehyde in PBS for 15 min at room temperature and UVirradiated during the fixation step. Cells were washed with PBS before blocked with 0.1 % (v/v) Triton
X-100 (Sigma-Aldrich)/ 5 % (v/v) FCS in PBS with blocking buffer for 45 min. Cells were then
incubated with primary antibodies diluted in antibody dilution buffer (0.05 % (v/v) Triton X- 100/ 5 %
(v/v) FCS in PBS) for 2 h and with the secondary antibodies for 1 h. Finally, cells were washed with
PBS and stained with Hoechst 33258 (Invitrogen). Cells were stored in PBS and fluorescence images
were captured by inverse optical microscopy.
IFN type I secretion following NDV infection. Biologically active interferon type I in cell culture
supernatants of NDV-infected cells was quantified using the iLite AlphaBeta human Type I activity
detection kit (Neutekbio; Galway, Ireland) according to the manufacturer’s instructions.
Cell survival experiment of IFN-stimulated and NDV-infected cells. RT3 K1 cells grown in 96wells were pretreated with IFNα or β (PeproTech; Hamburg, Germany) overnight. Subsequently, cells
were infected with NDV-EGFP or mock infected in the absence of IFN type I. Cell viability was
measured 72 h post infection using the CellTiter-Glo® Luminescent cell viability assay according to
manufacturer`s instructions (Promega).
Viral EGFP expression following IFNα treatment. HaCaT, RT3 and RT3 K1 cells grown in 96wells were pretreated with 0, 10 or 100 IU/ ml of IFNα overnight. Subsequently, cells were infected
with NDV-EGFP (MOI 0.01). Fluorescence images of viral EGFP expression were captured at 24 h
p.i.
Generation of NDV-specific siRNAs. NDV-specific siRNAs targeting the ORF of the P and V
mRNA (viral P+V siRNA) were designed and synthesized at Thermo Scientific Dharmacon (Chicago,
IL, USA).. #1: sense sequence: 5`-CGACAAGCUUAGCAAUAAAUU and antisense sequence: 5`PUUUAUUGCUAAGCUUGUC GU U; #2: sense sequence: 5`-GCGCAAUCCCACAAGGCAAUU
and antisense sequence: 5`-UUGCCUUGUGGGA UUGCGCUU; #3: sense sequence: 5`-GGACA
GAUCAGACAAACAAUU and antisense sequence: 5`-UUGUUUGUCUGAUCUGUCCUU.
siRNA library and single siRNAs ( Thermo Scientific Dharmacon). Human siARRAY RTF
Membrane Trafficking library (#H-005500) including siControl RISC-free siRNA (D-001220-01-05)
and a pool of NDV- specific siRNAs # 1-3;
H-Ras siRNA (LQ-004142-00), Rac1 siRNAs (D-003560-07, D-003560-08, D-003560-30), ONTARGETplus Non-Targeting Pool siRNAs (D-001810-10) and siGENOME Non-Targeting siRNA #3
(D-001210-03).
Reverse transfection of siRNA. SiRNA library transfections were performed according to the
manufacturer’s instructions using a 1.5 % (v/v) mastermix of DharmaFECT 3 transfection reagent
(Thermo Scientific Dharmacon) in Dharmafect cell culture reagent (DCCR) and 2x104 RT3 K1 cells.
For reverse transfection of single, reconstituted siRNAs, the mastermix including siRNA was preincubated before cells were added (final concentration of siRNA: 50 nM). HCT 116 cells were
transfected using a 1.32 % (v/v) DharmaFECT 1/ DCCR mastermix and 3x104 cells.
Screening of the siRNA library for NDV-sensitizing candidates. Transfected RT3 K1 cells were
infected with NDV-luciferase 48 h post transfection. Viral luciferase activity was measured 24 h post
infection. In parallel, transfected cells were mock infected to perform a cell viability assay
(alamarBlue® assay; Invitrogen) 72 h post transfection according to the manufacturer’s instructions.
Besides specific siRNA pools each screening plate contained an untreated control, a transfection only
control and a risc-free siRNA as negative controls. The positive control for inhibition of viral
replication comprised of the pooled viral P+V (1-3) siRNAs. SiRNAs which caused a reduction of cell
viability to more than 80 % compared to untreated cells were considered as cytotoxic.
cDNA synthesis. Cell lysis and subsequent cDNA synthesis without previous RNA isolation was
performed with the Cells-to-cDNA™ II Kit from Ambion (Austin, TX, USA) according to the
manufacturer’s instructions. 2x104 cells were lysed in 50 µl lysis buffer provided in the kit.
Realtime PCR Taqman analysis. Assays were run in a 7500 Fast Real-Time PCR System by ABI
Prism® (Applied Biosystems; Foster City, CA, USA). Singleplex reactions (in triplicate) were
performed using gene-specific pre-developed Taqman assays (PKR: Hs00169345_m1; OAS1:
Hs00242943_m1; MxA: Hs00182073_m1; Rac1: Hs01588892_g1; Rac1b: Hs01025984_m1) and predeveloped Taqman assays for endogenous controls (GAPDH #4333764 and 18s #4319413E) from
Applied Biosystems. Relative quantification of gene expression was performed by using the “Relative
Standard Curve Method”(Larionov et al., 2005).
Microscopy. Fluorescence and phase contrast images were captured using an inverse optical
microscope (Axiovert 25) and the Axio Vision Release 4.7.2.0 software (Carl Zeiss; Oberkochen,
Germany).