The sebaceous gland populations were generated from human scalp (SSG3), face, chest and breast from both male and female donors. The skin samples were collected as a surgical waste with information provided regarding the age and sex of the donors with Institutional Review Board (IRB) approval at Cincinnati Children’s Hospital Medical Center. Cincinnati Children’s Hospital is a Pediatric Hospital that allowed us to collect samples from donors ranging 9 months old to 12 years old. The IRB determined that the research does not meet the regulatory criteria for research involving human subjects as there were no interaction with the donors and no identifiable private information. After treating the skin with dispase overnight at 4°C, intact sebaceous glands were isolated with microsurgical instruments under a dissecting microscope (Figure 1b). To mimic the microenvironment of the sebaceous gland, the explants were sandwiched between glass coverslips coated with human fibronectin (10 μg/ml, Millipore, Billerica, MA) (Figure 1d). The explants were cultivated in sebocyte medium as described  (DMEM/Ham’s F-12 (3:1), Epidermal Growth Factor (EGF 3 ng/ml, Austral Biologicals, San Ramon, CA), cholera toxin (1.2×10-10M, Sigma, St. Louis, MO), adenine (24 μg/ml, Sigma, St. Louis, MO), insulin (10 ng/ml Sigma), hydrocortisone (45.2 ng/ml, Sigma), FBS (2.5% Hyclone, San Jose, CA), antibiotic/antimycotic (100×, Invitrogen, Grand Island, NY). After 1–2 weeks of growth in culture, cellular outgrowth became apparent from the periphery of the gland lobules. The explants were removed and the isolated cells cultured on the fibronectin-coated coverslips.
Proteins were separated by electrophoresis on 8-10% acrylamide gels, transferred to nitrocellulose membranes and subjected to immunoblotting. Membranes were blocked for one hour with 5% non-fat milk or 5% BSA in PBS containing 0.1% Tween-20. Primary antibodies were used at concentrations described below and HRP-coupled secondary antibodies were used at 1/2,000 in 5% non-fat milk. Immunoblots were developed using standard ECL (Amersham, Pittsburg, PA) and Luminata TM crescendo and classico (Millipore, Billerica, MA). Two-color immunoblot detection was performed using LI-COR Odyssey CL× (Biosciences, Lincoln, NE). Membranes were blocked in Odyssey blocking buffer (Li-Cor) and secondary antibodies conjugated to IRDye 680LT and 800CW were used (1/10,000; Li-Cor). Protein levels were quantified using the Odyssey Infrared Imaging System (Li-Cor).
To ablate TGFβ RII in SSG3 cells, we used shRNA vectors from the CCHMC Heart Institute lenti-shRNA library core (shRNA TGFβ RII #197031 and 194992 and a shRNA control). The human library was purchased from Sigma-Aldrich (MISSION shRNA). Lentivirus was produced by the Viral Vector Core at the Translational Core Laboratories, Cincinnati Children’s Hospital Research Foundation. Cells were grown to 80% confluency in 6-well plates before being infected with the lentivirus for 48 h. Infected cells were selected with 1 μg/ml puromycin (Sigma, St. Louis, MO) for 48 h. Following selection, TGFβ RII knock down cells were grown in regular media for 48 h before being activated with 5 ng/ml TGFβ1 for 24 h.
Histology and Immunofluorescence
Human tissues were frozen unfixed in OCT compound (Tissue-Tek, Sakura, Torrance, CA) for cryosectioning. Immunostainings were performed as previously described .
Primary antibodies against the following proteins were used at the dilution indicated: PPARγ (Santa-Cruz Biotechnology Inc., Santa Cruz, CA, H-100 1/250 for immunofluorescence, 1/500 for immunoblot), Blimp1 (Cell Signaling Technology, Beverly, MA, 1/500 for immunofluorescence, 1/1,000 for immunoblot), Fibronectin (Santa-Cruz Biotechnology Inc., Santa Cruz, CA, EP5 1/150), Muc1 (Millipore, Billerica, MA 1/500), cMyc (Cell Signaling Technology, Beverly, MA, 1/800 for immunofluorescence, 1/1,000 for immunoblot), TGFβ RII (Santa-Cruz Biotechnology Inc., Santa Cruz, CA, sc-220 1/1,000), p-Smad2 (Cell Signaling Technology, Beverly, MA, 1/100 for immunofluorescence, 1/1,000 for immunoblot), Smad2/3 (BD Biosciences, San Jose, CA, 1/500), α6 integrin (CD49f, BD Biosciences, San Jose, CA, 1/100), Keratin 8 (this antibody, developed by Dr. Brulet and Dr. Kemler, was obtained from the NICHD Developmental Studies Hybridoma Bank maintained by the University of Iowa, 1/1,000), β-actin (Sigma, St. Louis, MO, 1/2,000), Keratin 7 (Cell Signaling Technology, Beverly, MA, 1/1,000), 4′,6-diamidino-2-phenylindole (DAPI) was utilized as a marker of cell nuclei (Sigma Chemical Co., St. Louis, MO, 1/5,000). Secondary antibodies Alexa Fluor 488 or 555 (Molecular Probes, Grand Island, NY) were used at a dilution of 1/1,000. Fluorescence images were acquired with a fluorescent microscope AxioImager M1 (Zeiss, Oberkochen, Germany) and pictures were taken with an axioCam MRm camera (Zeiss, Oberkochen, Germany).
Total RNA was isolated using a Qiagen Rneasy Mini Kit and used to produce cDNA (Maxima first strand cDNA synthesis kit, Fermentas, San Jose, CA). Reverse transcription (RT) reactions were diluted to 10 ng/μl and 1μl of each RT was used for real-time PCR. Real-time PCR was performed using the CFX96 real-time PCR System, CFX Manager Software and the SsoFast EvaGreen Supermix reagents (Biorad, Hercules, CA). All reactions were run in triplicate and analyzed using the ΔΔCT method with relative expression normalized to GAPDH. Primers used:
GAPDH-F: ACATCGCTCAGACACCATG, GAPDH-R: TGTAGTTGAGGTCAATGAAGGG
PPARγ-F: GAGCCCAAGTTTGAGTTTGC, PPARγ-R: GCAGGTTGTCTTGAATGTCTTC,
FADS2-F: TGTCTACAGAAAACCCAAGTGG, FADS2-R: TGTGGAAGATGTTAGGCTTGG,
TGFβ RII-F: CTGTGGATGACCTGGCTAAC, TGFβ RII-R: CATTTCCCAGAGCACCAGAG
For Nile red staining, cells or OCT sections were fixed 10 minutes at room temperature in 4% formaldehyde. After 3 washes in 1XPBS, Nile red staining was performed with 0.1 μg/ml of Nile red (Sigma, St. Louis, MO) in 0.15 M NaCl for 15 minutes at room temperature. For Oil red O staining, cells were fixed 15 minutes in 10% formalin, wash with water for 10 minutes and 60% isopropanol before being stained with Oil red O (0.7% in 60% isopropanol) for 45 minutes. Cells were rinsed with 60% isopropanol and the nuclei stained with haematoxylin. To trigger differentiation of sebocytes in vitro, 0.1 mM linoleic acid (Sigma, St Louis, MO) was added directly to sebocyte media. To prepare cells for extraction of lipids, 2-3×107 of cells were pelleted, washed with 1XPBS and lipids were preserved in the dark at −80°C under argon until analysis. The qualitative and quantitative composition of lipids in scalp-derived human sebocytes was determined using an Agilent 5973N Gas chromatograph/Mass spectrometer with a SPE cartridge (solid phase extraction) and was performed by Synelvia S.A.S (Labege, France).
Nile Red analysis by FACS
Cells were cultured in 6-well plates at 80% confluence and infected with the lentivirus expressing the shRNAs as previously described. After puromycin selection for 48 h, cells were washed in 1X PBS and treated with working medium with or without Linoleic acid (0.1 mM) for 24 h. The cells were trypsinized, washed once with 1X PBS and neutral lipids were labeled with the fluorescent dye Nile red (1 μg/ml in PBS). 10,000 cells per sample were analyzed using a FACS Canto I equipped with a blue laser (488 nm excitation).
Cells were grown at 80% confluency in sebocyte media and rinsed once with 0.175 M sodium cacodylate buffer. Cells were fixed in 3% glutaraldehyde/0.175 M cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA) for 1 hour at 4°C. Dishes were washed twice with 0.175 M sodium cacodylate buffer. Cells were post fixed in 1% osmium tetroxide/cacodylate buffer for 1 hour at 4°C before being washed three times with 0.175 M sodium cacodylate buffer. After the final wash with 1.5 ml, cells were scraped and centrifuged for 5 min at 10,000 RPM. The cell pellet was then resuspended in 1 ml 1% agarose (Type IX ultra-low gelling tempt, Sigma) overnight at 4°C. The samples were then processed through a graded series of alcohols, infiltrated and embedded in LX-112 resin. After polymerization at 60°C for three days, ultrathin sections (100 nm) were cut using a Reichert-Jung Ultracut E microtome and counterstained in 2% aqueous uranyl acetate and Reynolds lead citrate. Images were taken with a transmission electron microscope (Hitachi H-6750) equipped with a digital camera (AMT 2k×2K tem CCD).
Data are expressed as means +/− SD. Comparison between two cell types was performed using unpaired two-tailed student’s t test. Paired two-tailed student’s t test was used when we compared the effect of a treatment on the same cell type. p<0.05 was considered significant.