Supplemental Figure 1. A) Untreated ears from C57BL/6 Dopachrome
tautomerase (DCT)-LacZ transgenic (gift of Ian Jackson) mice were stained for βgalactosidase30 to identify melanocytes. Shown are representative whole mount
sections (63x magnification) of animals of the indicated pigmentation phenotype.
Note that although pigmentation partially obscures blue stained (LacZ +)
melanocytes, cutaneous melanocytes are present at comparable densities in all
pigmentation groups. regardless of pigmentation phenotype. B) Media transfer
experiments (as in Fig. 1B) testing MITF induction in melanoma cells after
incubation with conditioned medium from UV irradiated melanocytes or direct
treatment with 30 µg/ml MSH (Sigma). C) Quantitative pcr-based detection of
POMC mRNA induction at 24h following UV radiation of Melan-c mouse
melanocytes or primary human melanocytes. PCR data are reported as fold
induction normalized to GAPDH (control) +/- standard deviation of at least three
samples per condition. D) Representative whole mount sections (63x
magnification) of K14-SCF DCT-LacZ compound transgenic animals of the
indicated pigmentation phenotype as described in (A).
Supplemental Figure 2. Photographs and melanin quantification of C57BL/6
pigment variants used in this study. A) photographs of 4-8 week-old K14-SCF
transgenic mice of the indicated genotypes. Genetic status of the tyrosinase
gene (Tyr) and melanocortin 1 receptor (MC1R) are indicated. B) Whole
depillated skin was collected from three 6-8 week-old K14-SCF transgenic
C57BL/6 animals of the indicated pigment phenotype and lyophilized before
HPLC-based quantification of pheomelanin (B) or eumelanin (C) as described 17
and presented as means +/- standard deviation of at least three samples from
different animals per condition . D) Photographs of depillated pheomelanotic
(mc1re/e) K14-SCF transgenic C57BL/6 mice treated topically with vehicle control
(70% ethanol, 30% propylene glycol), irradiated with 200 mJ/cm 2 UVB, or treated
topically with 400 µl of C. forskohlii root extract (80 µmoles forskolin) to the dorsal
skin surface for 21 days.
Supplemental Figure 3. A) Time and dose response study of forskolin-induced
skin darkening of mc1re/e K14-SCF+ animals treated as indicated. Data are
presented as means +/- SD of skin reflectometry measurement29 from at least
three animals per treatment group. Days that topical treatments began and
ended are indicated by the arrows. Of note, skin color measurements of
untreated K14-SCF transgenic animals on the MC1RE/E (black) background
(maximally melanized) averaged 21.5 +/- 1.8 on the CIE L* color axis. Therefore
forskolin-induced eumelanization promoted near-maximal levels of pigmentation
in the mc1re/e background. This experiment was repeated with similar results. B)
Side-by-side photographs of forskolin-treated depillated mc1re/e mice either
containing epidermal melanocytes (K14-SCF+) or lacking epidermal melanocytes
(K14-SCF-), treated as described in Figure 2. C) Photograph of groups of
pheomelanotic (mc1re/e) K14-SCF transgenic depillated mice treated with either
vehicle control or C. forskohlii root extract (20 moles forskolin) as indicated
applied only to the rump, for 21 days. Note that the darkening observed in two of
the control-treated mice represents re-growth of anagen hair rather than skin
melanization.
Supplemental Figure 4. A) Comparison of skin melanization induced by pure
forskolin vs. C. forskohlii root extract. The upper panel shows photographs of
representative mc1r e/e K14-SCF+ animals treated with 15 µl applied to the right
ear (see white triangles) of either vehicle control (left column), 1.5 moles HPLCpurified forskolin (middle column) or with 1.5 moles forskolin (as C. forskohlii
root extract (20% w/w forskolin; right column)) over a two week period. The
lower photographs depict Fontana-Masson (melanin) stained sections of treated
ears from the photographed animals. Note that the actual forskolin concentration
was the same in the crude preparation used as in the purified preparation. B)
Thymine dimer formation in the skin of depillated animals treated as described in
Figure 3 as detected by immunohistochemistry to control for the possibility that
melanin deposition could interfere with fluorescence-based thymine dimer
detection. Note that forskolin-pretreatment of fair-skinned animals provided
profound protection (as much as genetically black wild type animals) whereas
fair-skinned animals pre-treated with vehicle (control) exhibited almost as much
nuclear thymine dimer staining as amelanotic (albino) animals. C) Induction of
melanin by topical forskolin treatment does not depend on presence of the K14SCF transgene. Photographs in the left column depict ears of K14-SCF negative
MC1RE/E animals either untreated or subjected to daily application (5d/wk, 21
total treatments) with either vehicle control (70% ethanol, 30% propylene glycol)
or with forskolin (1.5 or 3 micromoles daily). Shown in the right column are
representative skin sections of corresponding ears treated as described and
stained by the Fontana-Masson method for melanin accumulation (630x).
Supplemental Figure 5. A) Forskolin treatment (80 µmoles) prevented UVinduced weight loss. After 16 weeks of UV treatment, vehicle/UV-irradiated
animals were significantly smaller than their forskolin/UV-irradiated counterparts.
B) Photographs of vehicle-treated or forskolin-treated depillated animals after 16
weeks of UV exposure as indicated. Note the smaller size and the many
ulcerative and hyperkeratotic skin changes in control/irradiated animals and no
evidence of lesions in forskolin/irradiated animals. We observed no side effects
of chronic forskolin treatment during the topical application course of this
experiment.
Supplemental Figure 6. Protective effect of topical forskolin against UV-induced
skin pathology in nucleotide excision repair-deficient mice. K14-SCF transgenic
mc1re/e depillated animals deficient for the XPC gene were treated as described
in Figure 4. A) Representative skin histological examination of vehicle control(left column) or forskolin-treated (right column) irradiated animals after 16 weeks
of treatment. Shown are Hematoxylin & Eosin (H-E) stained sections and
Fontana-Masson (F-M) stained sections (in which melanin appears as black) of
treated (dorsal skin; upper three panels) vs. untreated (ventral) skin on the same
animal. Note that vehicle control-treated irradiated animals had significant
epidermal (red bars) and dermal (yellow bars) thickening as compared to their
forskolin-treated irradiated counterparts. B) Graphical representation of the
epidermal thickness of groups of mc1re/e K14-SCF+ xpc-/- animals as labeled.
Data are expressed as mean per cent thickness +/- SD of forskolin-treated,
irradiated dorsal skin (three independent measurements per condition). Note
that differences in epidermal thickness were highly statistically significant
between exposed, irradiated vehicle-treated versus forskolin-treated skin
sections as compared to either non-UV-exposed ventral (belly) skin from the
same UV irradiated mice or forskolin-pretreated UV-exposed XPC null littermate
controls.