performed under isoflurane anesthesia, and all efforts were made to minimize suffering. Cell culture NHEMs were maintained in Medium 254 (Kurabo) supple- mented with 5 mg/ml insulin, 5 mg/ml transferrin, 3 ng/ml human recombinant basic fibroblast growth factor, 0.18 mg/ml hydrocortisone, 3 mg/ml heparin, 10 ng/ml phorbol 12-myristate 13-acetate (PMA), 0.2% (v/v) bovine pituitary extract (BPE), and 0.5% (v/v) fetal bovine serum (FBS) (Invitrogen) at 37uC in an atmosphere of 5% (v/v) CO2. NHEKs were maintained in Epilife medium (Kurabo) supplemented with 10 mg/ml insulin, 0.1 mg/ml human recombinant epidermal growth factor (EGF), 0.5 mg/ml hydrocortisone, 50 mg/ml gentamycin, 50 ng/ml amphotericin B, and 0.4% (v/v) BPE under the same conditions described above. Human skin fibroblasts were maintained in DMEM (Invitrogen) supplemented with 10% (v/v) FBS under the same conditions described above as well. Transmission electron microscopy (TEM) For TEM analysis, human skins and HSS samples were fixed with 1/2 strength Karnovsky’s fixation buffer, followed by 3 washes with 0.2 M sodium cacodylate buffer, and post-fixation with 1% osmium tetroxide containing 1.5% potassium ferrocya- nide. After dehydration, tissues were embedded in Spurr’s resin. Sections were obtained using a ULTRACUT S ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) and were stained with uranyl acetate and lead citrate. Sections were observed and selected images were digitally registered using a H-7650 transmis- sion electron microscope (Hitachi High-Tech, Tokyo, Japan). Grafting cells onto SCID mice HSSs were prepared on SCID mice as previously described [39]. In short, melanocytes (1.06106), keratinocytes (6.06106), and fibroblasts (6.06106) were collected using 0.1% trypsin/EDTA, neutralized with an equal volume of FBS and then mixed and centrifuged for re-suspension. Silicone chambers (Renner, Darm- stadt, Germany) were sutured onto the dorsal skins of mice and a suspension of combined cells was added into the 3 mm opening at the top of the chamber 3 hr after the installation of the chambers. One week after the implantation, the top part of each chamber was removed to increase air exposure and a wire net was attached to protect the HSSs from external damage. The remaining part of the chamber, including the base, was spontaneously released from the back of each mouse approximately four weeks after the implantation. Measurement of melanin content Melanocytes were seeded at a concentration of 2.06104/cm2 in melanocyte culture medium without PMA and were cultured for 3 days. Cells were washed three times with PBS and were then dissolved with 2 M NaOH. The HSSs were surgically removed and incubated in 2 M NaBr to separate the epidermis from the dermis. The epidermis was soaked in 150 ml 2 M NaOH until completely dissolved. The absorbance of melanin at 405 nm was measured with a Model 550 Microplate Reader (Bio-Rad, Hercules, CA), and a melanin standard curve was prepared using synthetic melanin (Sigma-Aldrich, St Louis, MO). For the cultured melanocytes, melanin levels were quantitated and normalized by the amount of protein. The melanin level of cell line 1 was relatively presented to be 1.0. For the HSSs, melanin levels were quantitated per area of a punch biopsy 3 mm in diameter and the melanin level of the darkest HSS was relatively shown as 1.0. Role of RAB27A in Skin Color Determination Tyrosinase activity measurement Melanocytes were seeded in 12-well culture dishes at a density of 16105 cells/ml in melanocyte culture medium without PMA. After two days in culture, the cells were labeled overnight with 1.0 mCi/ml [3H]tyrosine and a 400 ml portion of the medium was applied to a water-equilibrated Sep-Pak AC-2 cartridge (Waters, Milford, MA). The released 3H2O was then eluted from the cartridge with 8 ml water and the eluate was transferred to a vial and mixed with scintillation fluid. The radioactivity was determined using a scintillation counter and was normalized by the amount of total protein, then the enzyme activity of cell line 1 (Fig. 2E) was presented to be 1.0. Melanocyte-keratinocyte co-culture assay NHEKs were harvested at a concentration of 2.06104/cm2 with keratinocyte culture medium. After incubation for 24 hr, NHEMs were added at a concentration of 1.06104/cm2 in melanocyte culture medium without PMA and keratinocyte culture medium without human recombinant EGF and BPE at the ratio of 1:2, followed by co-culture for another 3 days. Cells were washed and then fixed with ice-cold methanol and used for routine immunohistochemistry. The human gp100 antibody, HMB45 (DAKO, Produktionsvej, Denmark) at a 1:40 dilution, was utilized for melanosome detection. Cell nuclei were stained with DAPI. Ten images were captured using a NIKON digital camera for each sample and the ratio of melanosome transfer was analyzed. Quantitative real time RT-PCR Each HSS was pretreated with RNAlater (Qiagen, Valencia, CA) to stabilize total RNA and then the epidermal sheet was removed by incubation in 2 M NaBr. Total RNA from melanocyte cell cultures and epidermal sheets from HSSs was extracted using an RNeasy micro kit (Qiagen), followed by cDNA synthesis by reverse transcription of total RNA using a High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). On-demand probes for human RAB27A, MITF and GAPDH in TaqMan Gene Expression Assays (Applied Biosystems) were used for real time quantitative RT-PCR performed in an ABI PRISM 7300 sequence detection system (Applied Biosystems). Western blotting analysis Cultured melanocytes were washed with PBS and then solubilized in RIPA buffer (Sigma). A sample (5 mg) was separated using 12% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) after conventional extraction. A polyclonal RAB27A antibody from Santa Cruz (diluted at 1:500), a monoclonal tyrosinase antibody from Upstate Biotechnology, Inc. (Lake Placid NY) (diluted at 1:1000) and a monoclonal b-actin antibody from Sigma-Aldrich, Inc. (diluted at 1:5000) were used as primary antibodies, followed by detection of the protein of interest using anti-mouse IgG horseradish peroxidase (HRP)-conjugated (GE Healthcare UK Ltd., Buckinghamshire, England) or anti-rabbit IgG HRP- conjugated (GE Healthcare UK Ltd.) as secondary antibodies. Vector design Three RAB27A targeted BLOCK-iTTM shRNA vector se- quences were designed and cloned into the pENTRTM/U6 vector supplied from Invitrogen. The efficacy of shRNA was evaluated using the Target Screening System (Invitrogen) and then the sequence with the highest efficacy in gene silencing was selected and cloned into the pLenti6/BLOCK-iT-DESTTM destination vector (Invitrogen). pENTRTM/U6-shRNA-Scramble Med GC PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e41160