JCS025502 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Table S1. Uniquely regulated genes between bulge and UI follicular keratinocytes. List of genes upregulated or downregulated (twofold or higher, Student’s t-test P<0.05) in both UI and bulge stem cells compared with all α6-positive cells. Lists contain known genes only.

• Supplemental Figure S1 -

  Fig. S1. Colony-forming and seeding efficiency of FACS-sorted epidermal keratinocytes. (A-B) All six FACS-sorted cell populations were plated at equal density as previously described and cultured for 2 weeks in keratinocyte serum-free medium. Cultures were stained with Rhodamine and the average (n=3 plates per group) total colony numbers (A) or the percentage of large colonies (>1 mm) (B) were quantified using NIH ImageJ software and statistically compared using a Student’s t-test (*P<0.05, statistically different compared with UI cells; error bars represent s.d.). (C) All six FACS-sorted cell populations were plated at equal density as previously described, in FAD medium for 8 hours, after which unattached cells were washed away and attached cells were trypsinized and counted. Data are represented as the average total number of attached cells per dish (n=3 plates per group).

• Supplemental Figure S2 -

  Fig. S2. Sca-1 and keratin K15 immunofluorescence. Dorsal skin cryosections (A) and epidermal whole mounts were prepared from tail skin (B) and labeled with antibodies against the cell surface marker Sca-1 (A) or cytokeratin K15 (B). Fluorescent images were taken with a Zeiss LSM 510 confocal microscope. K15 immunoreactivity is present in the bulge region but not in the UI region or IFD (B). Scale bar: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. FACS analysis of UI cells derived from ACTB-EGFP mice. Freshly isolated epidermal keratinocytes derived from ACTB-EGFP transgenic mice were labeled with antibodies against α6 integrin, Sca-1 and CD34. α6 integrin expression was detected with Alexa Fluor 680-conjugated secondary antibodies to circumvent EGFP fluorescence. Viable basal cells were identified (A,C) and further gated by their surface expression of CD34 and Sca-1 (B,D).

• Supplemental Figure S4 -

  Fig. S4. Hair reconstitution assay of FACS-sorted UI cells. (A-H) FACS-sorted keratinocyte populations isolated from FVB mouse skin (1.25×10⁶ cells/graft) were mixed with dermal fibroblasts and injected into silicon graft chambers. Chambers were removed 1 week after cell injection and images of reconstituted skin and hair were captured 6 weeks post grafting. Bottom panels represent a side view of each panel above. Dashed circle designates the graft perimeter (A-B,E-F,G-H). (I) Hematoxylin and eosin-stained histological section of UI-derived skin graft (C,D) showing intact IFE and HFs.

• Supplemental Figure S5 -

  Fig. S5. DNA analysis of FACS-sorted UI cells. FACS dot plot of EdU-positive cells from bulge (A), UI (B) and IFE + IFD (C) sorted populations isolated from 7-week-old mouse skin 16 hours following a single pulse of Edu. Edu-positive cells are partitioned within the red box in each plot.

• Supplemental Figure S6 -

  Fig. S6. Microarray analysis of UI keratinocytes. (A) Experimental design model comparing UI with bulge and all α6-positive populations. (B) Correlation coefficient heat map showing commonality with respect to the total pools of altered genes between replicates in each experimental group. (C) Venn diagrams showing genes upregulated or downregulated (twofold or higher, Student’s t-test P<0.05) in both UI and bulge stem cells compared with all α6-positive cells.