Supplement legend
SUPPL. TABLE S1. Predicted molecular weights of sFasL chimeric proteins. The
molecular weight (mw) of the monomeric form of the differing chimeric molecules was
predicted using Clone Manager. The multimeric weights were then derived by
multiplying the monomeric mw by the number of molecules in the specified multimeric
forms. Note this does not compensate for any glycosylation or other post translational
modifications and are therefore used as an approximation. All values are shown in
kilodaltons.
SUPPL. FIG. S1. COL domain protein sequence and codon optimized DNA coding
sequence. (a) Amino acid sequence for the protein domain representing the COL
domain in all constructs described. Protein COL domain architecture was previously
developed by Fan et al.. The domains shown are: the hinge region of human IgG
(underlined), collagen-like peptide repeat (GPP)10, and the NC1 domain of type XXI
collagen (italicized). (b) COL domain DNA sequence. This DNA sequence was codon
optimized for mammalian expression prior to being synthesized.
SUPPL. FIG. S2. Adjusted COL-sFasL levels. HCT116 cells were transfected with
GFP, COL-sFasL, and Her2-COL-sFasL constructs. Media was collected after 48 hours
and briefly centrifuged. COL-sFasL media was diluted 1:2, 1:5, 1:10, and 1:25 times
using GFP control media. Equal volumes of diluted medias were subjected to Western
blot analysis under reducing conditions and detected using HA tag antibody.
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Densitometric analysis results shown were quantified using ImageJ. 1:10 dilution used
in Fig 2C.
SUPPL. FIG. S3. Schematic illustration of virus construction. The schematic represents
the progression of alterations made from wild type HSV-2 to FusOn-H3-RFP, which was
used for the final virus construction in Figure 3. (a) The HSV-2 backbone is shown with
the wild type ICP10 locus enlarged. The ICP10 protein consists of the Ras activating
and ribonucleotide reductase (RR) domains represented by individual rectangles. (b)
The FusOn-H2 ICP10 locus is shown. FusOn-H2 has the green florescent protein (GFP)
coding sequence fused to the ICP10 RR domain (coding sequence for amino acids 4031142). Also, as depicted the ICP10 promoter is replaced by the cytomegalovirus (CMV)
promoter. (c) FusOn-H3, which was constructed by deleting the GFP coding sequence
from FusOn-H2. (d) FusOn-H3-RFP, which was used for the final virus construction in
Figure 3, was constructed by inserting a RFP gene cassette flanked by loxp and lox2272
sites, to the 5’ end of the modified ICP10 gene in FusOn-H3.
SUPPL. FIG. S4. Verification of sFasL chimeric molecule insertion into the recombinant
viruses. Viral DNA was collected from the multiple recombinant viruses. PCR analysis
was used to verify molecule insertion into the viral backbone. (a) An image of the gel
shows PCR products verifying insertion of the sFasL chimeric proteins. The primers
Secr-F (forward primer recognizing the secretion signal) and sFasL-R (reverse primer
recognizing the C terminal portion of sFasL) were used except for FusOn-H3-Her2-COL
virus that does not contain sFasL. Her2-COL was amplified using Secr-F and COL-R
(primer recognizing the C terminal portion of COL domain) primers. (b) Predicted
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amplified PCR product sizes. (---) no primer binding sites therefore no expected PCR
product.
SUPPL. FIG. S5. Verification of sFasL chimeric molecule insertion at the correct RMCE
locus. Viral DNA was collected from the multiple recombinant viruses. PCR analysis
was used to verify molecule insertion between the lox2272 and loxp sites. (a) A
schematic of the primer binding locations. PK Rev 1, forward primer; PK Rev 2, reverse
primer; ICP10, infected cell protein 10 gene; loxp and lox2272, cre recombinase binding
sites; SV40P, SV40 promoter; RFP, red fluorescent protein gene; SV40pA, SV40
polyadenylation sequence; CMV, cytomegalovirus promoter; RR1, ribonucleotide
reductase carboxyl terminus. (b) An image of the gel shows PCR products verifying
insertion at the correct locus. (c) Predicted amplified PCR product sizes for the
respective viruses are listed in kilobases.
SUPPL. FIG. S6. Reduced 4T1 cell viability following supernatant transfer. HCT116
cells were transfected with respective sFasL molecules. Media was collected after 48
hours and cellular debris removed. Triplicate wells of 4T1 cells were treated with an
equal volume of each media. After 48 hours, viable 4T1 cells were quantified using the
trypan blue exclusion assay. The triplicate values were then averaged and percent
viable cells calculated by normalization to GFP control media treated cells. ** p<0.001 as
compared with GFP transfected cell media treatment controls using student’s T test.
Data representative of three independent experiments. Graph shows mean ±SEM.
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