Khaled_Fig. S MITF TYROSINASE R²= MITF PDE4D R²= Variance from mean mrna expression

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1 Khaled_Fig. S Variance from mean mrna expression.8 2 MITF.6 TYROSINASE.4 R²= MALME3M SKMEL28 UACC257 MITF PDE4D R²= MITF PDE4B R²= SKMEL2 SKMEL5 UACC62 M4 MITF PDE4C R²=-.44 MDAMB435 Figure S PDE4D and PDE4B mrna expression correlate with MITF expression. Quantitative mrna levels for each gene in eight human melanomas were acquired from Affymetrix microarrays as described by (Du et al. 24).

2 Khaled_Fig. S2 sictrl si MITF MITF Tub Figure S2 sirna mediated knock down of MITF. Human primary melanocytes were transfected 3 times with simitf. 72h after the first transfection, protein extracts were immunoblotted using antibodies against MITF (C5).

3 exons Khaled_Fig. S3 Nterm. Unique region PDE4D 5 UTR ucr ucr2 Catalytic domain C-term and 3 UTR D3 5 UTR D3 5 UTR Long isoforms D4 5 UTR D7 5 UTR D8 5 UTR Figure S4 schematic representation of PDE4D isoforms mrna structure. All PDE4D isoforms harbor a unique N- terminal region and are divided in three categories according to their length. The positions of the primers used in the study are displayed of the scheme. The scale of the length of each N-terminal region and of the length of each domain is not respected. D9 5 UTR D 5 UTR Short isoforms D2 5 UTR Super short isoform D6 5 UTR Pan isoforms PDE4D primers PCR primers qpcr primers

4 A Khaled_Fig. S4 53bp PDE4D 455bp PDE4D2 29bp PDE4D3 347bp PDE4D4 2bp PDE4D5 24bp PDE4D6 6bp PDE4D7 226bp PDE4D8 475bp PDE4D9 49bp NHM 5 heart Sk-n-sh B-Actin B Melanocytes Relative mrna expression 3 2 PDE4D PDE4D2 PDE4D3 PDE4D5 PDE4D6 PDE4D7 control fsk 2h fsk 6h fsk 2h PDE4D9 Figure S3 PDE4D isoform profiling. (A) cdna from sk-n-sh, cells, and 5 different primary human melanocytes (NHM) cultures was assessed by PCR. (B) Total mrna from normal human melanocytes exposed to 2 µm (fsk) was subjected to qpcr. The data are normalized to b-actin and each point is the mean ± SD of three experiments performed in duplicate. () P<.5.

5 Khaled_Fig. S5 A Relative mrna expression,4,2,8,6,4,2 Fibroblasts PDE4D PDE4D3 cont 2h 6h 2h B Fiborblasts PCREB tub 2h 6h 2h Figure S5 camp does not stimulate PDE4D or PDE4D3 mrna expression in normal human fibroblasts. (A) Total mrna of normal human fibroblasts exposed to 2 µm was subjected to qpcr. The data are normalized relative to b-actin and each point is the mean ± SD of three experiments performed in duplicate. (B) Extracts from primary human fibroblasts treated with as indicated were immunoblotted using antibodies against phospho-creb and a-tubulin.

6 Khaled_Fig. S6 A MITF si #2MITF sictrl simitf 2.5 cont 2h 6h 2h PDE4D cont 2h 6h 2h B MITF TUB cont 2h 6h 2h sictrl cont 2h 6h 2h simitf Figure S6 camp-induced upregulation of PDE4D3 is dependent upon MITF. (A) Primary human melanocytes were transfected with a non targeting sirna (sictrl) or sirnas targeting MITF and exposed to as indicated. Total mrna was subjected to qpcr. Results are expressed as fold stimulation and represent the mean ± SD of three independent experiments. () p<.5. (B) primary human melanocytes were transfected with simitf and treated as indicated. Protein extracts were immunoblotted using antibodies against MITF and a-tubulin.

7 + Nuclear extract Mouse IgG + αmitf-ab Competitor Wt Mut Khaled_Fig. S7 MITF supershift Free probe Figure S7 MITF Binds the PDE4D3 Ebox in vitro. Probe containing the Ebox from the human PDE4D3 promoter was used in EMSA to test in vitro binding of MITF to this sequence. UACC 257 melanoma nuclear extracts were used as a source of MITF, and a monoclonal anti-mitf antibody was used for supershift analysis. Supershifted complexe was observed on addition of wild-type biotinilated Ebox Wt probe in the presence of anti-mitf antibody. The supershifted band was competed away with increasing amounts of wild-type unlabeled probe but not with identical amounts of point-mutated unlabeled probe.

8 Khaled_Fig. S8 A Absorbance, variance from mean.5.5 control rolipram +rolipram 24h 48h 72h 96h B Cont Figure S8 Rolipram does not affect human primary melanocyte proliferation and morphology. (A) Human primary melanocyte were seeded in 96 well plate and treated as indicated. Every 24 h for 96h, WST- reagent was added to three well per condition and read at 44 nm 8 h after addition of the reagent. (B) Normal melanoma cells were treated as indicated for 48h. The pictures were taken at 4x magnification. rolipram Forskolin +rolipram

9 Khaled_Fig. S9.25 Pde4d sictrl si#pde4d3 si#2pde4d3 Figure S9 Efficiency of PDE4D sirna. Primary human melanocytes were transfected with a non targeting sirna (si cont) or a sirna specific for PDE4D. Total mrna was subjected to qpcr. The results are normalized relative to b-actin. () P<.5. Results are normalized relative to b-actin. Each data point is the mean ± SD of three experiments.

10 A Pde4d sictrl sipde4d3 DMSO PMEL7 LEF Day DMSO Day 2 t, 8h 8h B sictrl si PDE4d3 si PDE4d5 sipde4d3 sipde4d5 Pde4d sictrl + - sipde4d5 DMSO + - sipde4d h 8h 8h 8h sipde4d3 Khaled_Fig. S Figure S Forskolin pretreatment induces resistance that is reversible by PDE4D3 knockdown but not by PDE4D5 knockdown. (A) Primary human melanocytes were transfected with a non-targeting sirna (sictrl), sirnas targeting PDE4D3 or sirnas targeting PDE4D5. 24h after transfection, the cells were placed in minimal media for 4h and subsequently treated with for 4h.before being returned to minimal media. The following day, the cells were exposed to for the indicated times. Total mrna was subjected to qpcr. The results are normalized relative to b-actin. () p<.5. (B). Total mrna from experiments presented on panel (A) were subjected to qpcr. The results are normalized relative to b- actin. Each data point is the mean ± SD of three experiments.