on a Leica TCS SP5 confocal microscope. Z-stacks were acquired at 100 Hz with an optimal stack distance and 102461024 dpi resolutions. Z-stack projections were generated using the LAS AF software package (Leica Microsystems). All the images were processed with Photoshop CS4 (Adobe) software. Protein extraction and Western blotting Proteins were extracted from cultured keratinocytes or total skin. 1 cm2 pieces of total back skin (dermis and epidermis) were snap-frozen in liquid N2, transferred to lysis buffer (1% NP-40, 200 mM NaCl, 25 mM Tris-HCl, pH 8, 1 mM DTT) including protease inhibitor cocktail (Roche) and homogenised for 30 sec- onds. Samples were incubated on ice for 20 minutes. Protein lysates were cleared by centrifugation at 13,000 rpm. Total protein concentration was quantified using Dc Protein Assay (Bio-Rad). Equal amounts of protein were run in 7.5% polyacrylamide gels and blotted onto Hybond-P PVDF mem- branes (GE Healthcare), which were incubated in TBST-blocking solution (Tris-buffered saline, pH 8.8, with 5% skimmed milk powder). Blots were incubated overnight at 4uC with primary antibodies, washed and incubated with the appropriate HRP- conjugated secondary antibodies (GE Healthcare). a-Tubulin (Sigma) was used as a loading control. The chemiluminescent signal was detected using the ECL Plus Detection System (GE Healthcare). RNA extraction and quantitative RT–PCR (QPCR) Total RNA from mouse skin or cultured keratinocytes was prepared using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA from flow-sorted cells was purified using Pure-Link RNA Micro Isolation Kit (Invitro- gen). Double-stranded cDNA was generated from 1 mg total RNA using Superscript III First-Strand Synthesis kit (Invitrogen) and random hexamer primers (Promega). A minimum of two independent biological and three technical replicates was analysed. In case of Itga´6/CD34+ve-sorted cells one sample was pooled from four mice. For FDG-sorted cells one sample was pooled from one mouse. Real-time PCR amplification and analysis was conducted using the 7900HT Real-Time PCR System (Applied Biosystems). The standard amplification protocol was used with pre-designed probe sets and TaqMan Fast Universal PCR Master Mix (26) (Applied Biosystems). Probe set Mm00520224_m1 and Mm00487803_m1 were used to amplify mouse NSun2 (Misu) and c-Myc from total skin. The following probes were used to amplify selected genes from flow-sorted cells: a6 integrin (Mm01333831_m1), FGF-18 (Mm00433286_m1), CD34 (Mm00519283_m1), NFATc1 (Mm00479445_m1), Sox9 (Mm00448840_m1), Lgr5 (Mm00438890_m1), Sox6 (Mm00488393_ m1), Wnt5a (Mm00437347_m1), Lef1 (Mm00550265_m1), Gata3 (Mm01337569_m1) and Keratin 72 (Mm00495207_m1). GAPDH expression (4352932E) was used to normalize samples using the DCt method. Cloning, cell culture, transfection, and reporter assays The 22068 to 248 bp DNA fragment of the mouse Misu promoter was cloned into the pGL3-Basic vector (Promega). Plasmids for the reporter assays included pCDNA 3.1-hLef1-V5 (Lef1), pCDNA 3.1-DN63-hLef1-V5 (DLef1), and pCDNA 3.1- S33Y mCTNNB1 (b-catenin). pLef7-fos-luc (pLef7) was kindly provided by R. Grosschedl [52]. Hela cells were grown in DMEM (Invitrogen) supplemented with 10% fetal calf serum (FCS) in a humidified atmosphere at 37uC and 5% CO2. Cells were transiently co-transfected with the promoter construct, pRL-TK Misu Regulates Stem Cell Fate renilla as an internal control and the indicated plasmids using Lipofectamine LTX transfection reagent (Invitrogen). After recovery, cells were grown in media containing 0.2%FCS. Luciferase activity was measured 36–48 hours after the transfec- tion using the Dual-Luciferase Reporter Assay System (Promega) on Glomax (Promega). Each transfection was carried out in triplicate and the experiment was repeated twice. Flow cytometry, cell sorting, and cell cycle analysis To isolate mouse keratinocytes from dorsal back skin we rinsed mouse back skin in 10% Betadine and 70% ethanol and washed it in PBS. The dermal side was thoroughly scraped to remove excess fat. The tissue was then floated on 0.25% Trypsin without EDTA (Invitrogen) for 2 hours at 37uC or overnight at 4uC. The epidermis was subsequently scraped from the dermis, minced using scalpels, disaggregated by gentle pipetting and filtered through a 70 `ım cell strainer. Trypsin was inactivated by addition of low-calcium medium with 10% FCS. The cells were pelleted and resuspended in the following antibodies: PE-conjugated Itga´6 (clone GoH3, eBiosciences), Alexa Fluor 647-conjugated CD34 (RAM34, eBiosciences) and goat polyclonal anti-P-cadherin (R & D Systems). After incubation for 45 minutes at 4uC, cells were washed twice in PBS. For detection of P-cadherin, cells were incubated for 10 minutes at 4uC with anti-goat Alexa Fluor 647- congugated secondary antibody (Invitrogen). Cells were gated using forward versus side scatter to eliminate debris. Doublet discrimination was carried out using pulse width. The viable cells were then gated by their exclusion of DAPI using a 450/65 nm filter. Itga´6 PE stained cells were detected using a 580/30 nm filter and Alexa 647 cells were detected using a 670/ 30 nm filter. Cells were sorted with a MoFlo high-speed sorter (Beckman Coulter). For cell cycle analysis of Itga´6high/CD34+ve or Itga´6low/P- cadherinhigh cell populations, cells were fixed with 1% parafor- maldehyde for 5 minutes after immunolabelling, transferred to cold 70% ethanol and incubated for at least 1 hour before stained with propidium iodide (PI) or DAPI (Sigma). After incubating the cells for 1 hour in RNase, analysis was carried out on a CyAN ADP analyzer (Beckman Coulter). For detection of intracellular b-galactosidase activity, mouse keratinocytes from mice in early anagen (P21) were loaded with fluorescein-di-b-D-galactopyranoside (FDG) (Sigma) using hypo- tonic shock. Briefly, equal volume of cells was mixed with warm 26 hypothonic shock solution (2 mM FDG in water) and incubated for 30 seconds at 37uC, then cold media was added. Cells were washed and subsequently stained with the appropriate antibodies. Cells were then sorted based on FDG (fluorescence detected using a 530/40 nm filter), after gating out dead cells (based on DAPI staining). Cell culture of mouse keratinocytes, clonal growth, and differentiation assay Mouse keratinocytes were isolated as described above and cultured on mitomycin-treated J2-3T3 feeder cells on collagen type I (BD Biosciences) coated plates (BD Falcon). Mouse keratinocytes were grown in low-calcium FAD media (one part Ham’s F12, three parts Dulbecco’s modified Eagle’s medium, 18 mM adenine and 0.05 mM calcium) supplemented with 10% FCS and a cocktail of 0.5 mg/ml of hydrocortisone, 5 mg/ml insulin, 10210 M cholera enterotoxin, and 10 ng/ml epidermal growth factor (HICE cocktail) and maintained in a humidified atmosphere at 32uC and 8% CO2. Clonal growth was assayed by culturing 500 to 2500 Itga´6high/ CD34+ve sorted cells or 5000 to 10000 viable epidermal cells per PLoS Genetics | www.plosgenetics.org 11 December 2011 | Volume 7 | Issue 12 | e1002403