Supplemental Methods
Quantification of VOPP1 protein expression using ImageJ software (NIH)
Digital images of the western blots from tissue samples and cell lines were captured
and subsequently loaded into the ImageJ software. Using the Analyze  Gels set of
functions, which allows for background correction, VOPP1 specific bands were
assessed along with / tubulin. The ratio of VOPP1 signal to / tubulin per
sample was then constructed and served as an index of VOPP1 expression. In all
blots, the SCC-9 cell line was included and served as the standard reference. For
presentation purposes, the above described index of VOPP1 expression was
converted to fold change relative to benign squamous mucosa. The following
hypothetical data shows exactly how we derived protein expression levels of VOPP1
in terms of fold change from BSM.
Blot “A”
tissue
Blot “B”
Cell line
Cell Line
BSM
SCC
SCC-9
FaDu
HeLa
SCC-9
VOPP1
1465
4533
2634
5234
3123
3412
/ tubulin
ratio
1234
1056
783
543
432
452
1.19
4.30
3.36
9.64
7.23
7.55
1.28
0.96
1.00
3.62
2.72
2.83
relative to SCC-9
relative to BSM
1.00
3.62
2.83
Quantification from Invitrogen LIVE/DEAD® assay using ImageJ software (NIH)
Digital images captured from fluorescence microscopy of the assay were processed
in ImageJ in the following manner: (1) Using Process  Subtract Background,
background corrections were made. (2) Using Image  Adjust  Threshold, a
constant threshold level was set across a set of conditions to ensure that enough
area of signal was present to identify viable or dead cells (3) Using Process  Binary
 Watershed, a segmentation analyses was performed to allow for adequate
automated tabulation (4) Using Analyze  Analyze Particles a constant area
exclusion filter was applied to ensure that tiny specs of signal, likely from debris or
instrument artifact, were excluded from the final counts of live or dead cells. A
resulting index of cell death was constructed from the ratio of the number of dead
cell events divided by live cell events, which was expressed as fold change relative
to the control siRNA condition.
Cloning details of the construction of luciferase / renilla reporter system employed
pGL3-Con-hRluc (constitutively active renilla reporter)
The pGL3-Control vector (Promega) was digested with NcoI & XbaI and the
resulting 3604 base pair fragment representing the vector backbone was purified.
The pGL4.73 vector (Promega) harboring the hRluc reporter was also digested with
NcoI & XbaI and the 943 base pair fragment containing hRluc was purified. The two
fragments identified were then ligated in standard fashion and ultimately confirmed
via restriction digest mapping.
pGL3-minP-luc2 (minimal promoter luciferase reporter)
The pGL3-Control vector (Promega) was digested with NcoI & XbaI and the
resulting 3358 base pair fragment representing the vector backbone was purified.
The pGL4.20 vector (Promega) harboring the luc2 reporter was also digested with
NcoI & XbaI and the 1663 base pair fragment containing hRluc was purified. The two
fragments identified were then ligated in standard fashion and ultimately confirmed
via restriction digest mapping.
pGL3-NFB-luc2 (5X NFB & minimal promoter luciferase reporter)
The pNF-κB-Luc vector (Stratagene) was digested first with XbaI and a resulting
3945 base pair fragment was purified. This restriction fragment was further
digested with BamHI and the resulting 96 base pair fragment containing the 5X
NFκB element was purified. The pGL3-minP-luc2 vector described above was
digested with NheI & BglII and the resulting 5002 base pair fragment basically
representing the open vector was purified. The two fragments identified were
ligated in standard fashion and ultimately confirmed via restriction digest mapping.