Supplementary Information

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1 1 Supplementary Information Human TSC2 Null Fibroblast-Like Cells Induce Hair Follicle Neogenesis and Hamartoma Morphogenesis Shaowei Li 1, Rajesh L. Thangapazham 1, Ji-an Wang 1, Sangeetha Rajesh 1, Tzu-Cheg Kao 2, Leonard Sperling 1, Joel Moss 3, & Thomas N. Darling 1 1 Department of Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA 2 Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA and the 3 Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA

2 2 Supplementary Figures Supplementary Figure S1. Xenograft model for TSC skin hamartomas. Fibroblast-like cells from a skin hamartoma and neonatal foreskin keratinocytes are incorporated into a dermal-epidermal composite that is grafted onto the back of a nude mouse. The schematic shows hair follicle microanatomy that develops in grafts constructed with TSC2-null fibroblast-like cells. Hamartomatous features of tumour grafts are not shown, including increased angiogenesis and mononuclear phagocytes.

3 Supplementary Figure S2. Characterization of hair follicles in grafts with TSC2-null cells from an angiofibroma. TSC2-null fibroblast-like cells from an angiofibroma and neonatal foreskin keratinocytes were incorporated into dermalepidermal composites and grafted onto mice. (a) Cells in the dermis, epidermis, and hair follicles are reactive with a pan-human HLA class I monoclonal antibody. Scale bar, 65 µm. (b) Frozen sections of grafts were hybridized with a fluorescent probe of human Y chromosome. Red signals are observed in the nuclei of epidermal and follicular epithelial cells, but no signals are observed in dermal fibroblast-like cells from a female patient. Scale bar, 65 µm. 3

4 Supplementary Figure S3. Histological and immunohistological differences between TSC fibrous plaques and normal-appearing skin. (a) H & E stained section of TSC normal skin. (b) H & E stained section of fibrous forehead plaque. (c) In TSC normal skin, immunoreactivity for ps6 is faint and located mostly in the upper layers of the epidermis. (d) In fibrous plaques, immunoreactivity for ps6 is located in dermal fibroblast-like cells and the overlying epidermis. (e) In TSC normal skin, scattered cells in the basal layer of the epidermis are immunoreactive with Ki-67. (f) In fibrous plaques, the cells immunoreactive with Ki-67 are almost confluent in the basal layer of the epidermis. (g) In TSC normal skin, scattered cells in the dermis are immunoreactive with anti-cd68 antibodies, a marker of mononuclear phagocytes. (h) In fibrous plaques, immunoreactive cells are frequent in the dermis. (i) In TSC normal skin, CD-31 positive vessels are typically small in size and few in number. (j) In fibrous plaques, CD-31 positive vessels are prominent in number and size. The chromagen used for immunohistochemical studies was Vector Red, except Ki-67 staining, which used DAB (brown). Scale bar, 35 µm. Similar results were obtained using fibrous plaques from 4 patients. 4

5 Supplementary Figure S4. Presence of human cells in xenografts following 12 weeks treatment with rapamycin. (a) HLA immunostaining of xenografts containing fibroblasts or TSC2-null cells from patient 4 demonstrates human cells in epidermis and dermis of xenografts. NV, TSC normal fibroblasts with vehicle treatment; NR, TSC normal fibroblasts with rapamycin treatment; TV, TSC2-null cells with vehicle treatment; TR, TSC2-null cells with rapamycin treatment. Scale bar, 35 µm. (b) HLA positivity was quantified as fluorescence intensity in the dermis relative to the dermal area. Numbers of mice were 13 for NV, 14 for NR, 12 for TV, and 15 for TR. Results are mean ± SE. **p<

6 6 Supplementary Table Supplementary Table S1. Dermal Papilla Genes (DPG) overexpressed by fibroblast-like cells from angiofibromas at levels twofold or more those in fibroblasts from patients normal-appearing skin. DPG1 # DPG2 ## DPG1&2 ### CD302 TCEAL1 ABCA1 GALNT7 RPS6KA5 ASPH CDH11 TMEM100 ACTG2 GNAQ RUNX1 CRABP2 DPYD TMEM2 ACTN1 GNL2 RUNX2 CTBP2 DRAM1 TRPS1 ANKRD28 HCK RUNX3 FGFR1 FGFR2 TWIST1 APPL2 HDAC6 SLC1A1 GFRA1 FST VCAM1 ARID4B HSD17B7 SLC5A3 IGFBP3 GRB14 B3GNT2 IGFBP4 SLC6A6 LTBP1 LEPR B4GALT6 JAG1 SMS NDP LPAR1 BNIP2 JAK2 SOX11 PDGFRA MAGED2 C5ORF30 KAZ SPSB1 PRSS12 MDK C7ORF23 KIF5A TENC1 RGNEF MYST4 CAMK2N1 KLHDC2 TMOD3 SHOX2 NAGA CDC14B NID2 TNN SLC16A2 PAPPA CLCF1 NPTX2 TRIM24 TGFBR1 PBX1 COL8A2 NTRK3 UBE2M WNT5A PTGER3 CRYBG3 OMD VEGFA PTGFR DAAM2 PER2 ZBTB11 RAB40B DMXL2 PHF3 ZMYM5 RSRC1 EIF2C1 PLAG1 ZSCAN18 SERPINF1 ELAVL1 PPM1A SH2B2 EPHB6 PRKAR1B SH3BP5 ETNK1 PRRX1 SPOCK1 ETS1 QKI SSBP2 FAM49B RC3H2 STEAP1 FLRT2 RIOK2 # Genes overexpressed by angiofibroma cells that are common with DPGs from Rendl et al ## Genes overexpressed by angiofibroma cells that are common with DPGs from Driskell et al ### Genes overexpressed by angiofibroma cells that are common with DPGs from both Rendl et al and Driskell et al Genes are listed alphabetically. Bold font indicates p <0.05 for two tailed t-test comparing expression in angiofibroma cells to paired patient fibroblasts.