Dajee and Lazarov et al
Legends for Supplementary Figures
Supplementary Table I. Summary of clinical and histologic findings with regenerated
human skin tissue grafted to immune deficient mice.
Supplementary Table II. Summary of tumor formation in mice after subcutaneous
injection of human keratinocytes expressing Ras and NF-B subunits or Ras and IB.
Supplementary Fig. 1. Features of Ras-IBepidermal tumorigenesis. (A) Failure to
rescue Ras growth inhibition by all 5 active NF-B subunits. Proliferation of primary
human epidermal cells transduced with the vectors noted was determined, with cell
numbers shown at 72 hours after transduction. Data represents triplicate independent
transductions for each vector combination +/-S.D. (B) Appearance of human skin
regenerated on scid mice from primary human epidermal cells expressing either marker
gene transduction control or both oncogenic H-RasG12V (Ras) and IBM. Each image
represents an independently transduced human tissue specimen. (C) p65 immunostain of
skin tissue from E16.5 RelA+/+ and RelA-/- mice. Note nuclear distribution of p65 protein
(brown) in epidermis of wild-type mice and lack of detection in RelA-/- tissue; counterstaining is with methyl green. (D) Expression of H-Ras and phosphorylated active
ERK1/ERK2 (MAPK*) protein in regenerated epidermal tissue expressing either Ras-IB
or marker control compared to normal patient skin (NL) and a spontaneously arising SCC
(patient SCC; data representative of SCC tissue from 4 unrelated patients).
Supplementary Fig. 2. CDK4 mRNA expression as a function of p65 activation. CDK4
mRNA levels are unchanged in response to p65 activation. Northern blotting is shown
with CDK4 mRNA levels at a series of timepoints after expression of active p65,
normalized and quantitated to -actin loading control on the same lane.
Supplementary Fig. 3. Cell proliferation and hTERT protein expression. (A) CDK4
rescues epidermal growth inhibition by oncogenic Ras. Proliferation of primary human
epidermal cells transduced with the vectors noted was determined, with cell numbers
shown at 96 hours after transduction. Data represents triplicate independent
transductions for each vector combination +/-S.D. Study of the functional and mechanistic
aspects of Ras interactions with CDK4 are beyond the size constraints of this manuscript
and represent the focus of another body of work. (B) Expression of hTERT protein in
human epidermal tissue expressing Ras-IBand LacZ control at 8 weeks in vivo.
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Dajee and Lazarov et al
Immunoblots are shown, blots were stripped and re-probed with antibodies to -actin.
Optical densitometric quantitation normalized to actin control is shown below each hTERT
band; control hTERT level was assigned a relative value of 1.0. (C) Circulating antibodies
to laminin 5 [Sera 198] and 64 integrin [Sera 578].
Supplementary Fig. 4. Verification of full-length protein expression after retroviral gene
transfer. 18 hours following retroviral gene transfer, extracts were prepared from wild-type
and mutant primary human epidermal cells and immunoblotted with antibodies to the
introduced gene products. All blots were stripped and reprobed with antibody to -actin
control shown in bottom panels to assure equal loading, transfer and extract quality. (A)
Expression of H-RasG12V and IBM in primary keratinocytes from normal patients. Note
that the stabilized IBM mutant migrates lower on the gel than endogenous IB due to
deletion of C-terminal PEST sequences. Cells were serially transduced with vectors noted
at the top of the panels and immunoblotted with antibodies noted at the left of each panel.
(B) Expression of H-RasG12V, IBM, laminin 5 3 and 4 integrin in primary keratinocytes
from genetically characterized patients with either laminin 5 3-deficient [left panel] or
64 integrin-deficient [right panel] junctional epidermolysis bullosa. Cells were serially
transduced with vectors noted at the top of the panels [laminin 5 3 expression
vector=Lam5 and 4 integrin subunit expression vector=4] and immunoblotted with
antibodies noted at the left of each panel.
Supplementary Fig. 5. Ras and IB expressing tumor cells display a normal diploid
karyotype and lack evidence of chromosomal translocations. Cells were isolated from
subcutaneous and pulmonary tumors after 4 weeks of growth in vivo and karyotype
analysis was performed, representative data shown. (A) Giemsa banding (B) Spectral
karyotype analysis with fluorescence in situ hybridization (SKY-FISH).
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