DEV02868 Supplementary Material

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• Supplemental Table S1-S2 -

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• Supplemental Figure S1 -

  Fig. S1. p53 does not contribute to the Notch1-deficient phenotype in skin. Histological (H&E staining) and stereoscopic view of the skin from the specified genotypes. Note that p53 loss did not rescue the Notch1-deficient follicular phenotype. Scale bars: 200 μm.

• Supplemental Figure S2 -

  Fig. S2. mRNA expression patterns of TGFβ and IGF signaling components. TGFβ1 is expressed in hair matrix whereas TGFβ2 is only expressed in proximal hair matrix. TGFβ receptors are found in the matrix as well as the dermal papilla (arrows). Igfbp2 and Igfbp3 are found in dermal fibrobalsts and epidermal basal keratinocytes, respectively. Scale bars: 50μm.

• Supplemental Figure S3 -

  Fig. S3. TGFβ signaling in epithelial cells does not participate in the Notch1-deficient phenotype. (A) Histological (H&E staining) and stereoscopic view of the genotypes specified. Note that no observable difference between the presence and absence of Tgfbr2. Scale bars: 200μm. (B) Semi-quantitative PCR of genomic DNA from plucked hairs from wild-type, anagen-deleted and embryo-deleted Msx2-Cre;Notch1^flox/flox;Tgfbr2^flox/flox (anN1;TbR2 and emN1;TbR2, respectively) using primers specific for Notch1 and Tgfbr2 floxed alleles. Nrarp primer sets were used as a positive control. Note the absence of Tgfbr2 floxed allele in emN1;TbR2 hairs.

• Supplemental Figure S4 -

  Fig. S4. Cell non-autonomous role of Notch1 in maintaining melanocyte population in hair follicles. Immunohistochemical staining of wild-type, Msx2Cre;Rosa26R/+ (albino) and Msx2Cre;Notch1^flox/flox;Rosa26R/+ with Dct-1 (green) and β-galactosidase (lacZ, red) antibodies. Nuclei were stained with DAPI. Note that melanocytes (Dct-1-positive) do not show β-galactosidase staining and that the number of melanocytes in Notch1-deficient hair follicles is reduced. Scale bar: 50μm.

• Supplemental Figure S5 -

  Fig. S5. Igfbp3 mRNA expression in P9 emN1 and P16 wild-type follicles. Non-radioactive mRNA in situ hybridization with an Igfbp3 probe on postnatal day (P)9 Notch1-deficient mouse and on P16 wild-type skins. (C,D) Magnified views of the follicles in A and B, respectively. Note that P9 emN1 sections (A,C) were developed for 2 days wheras P16 wild-type sections were developed for 3 hours. Scale bars: 200 μm in A,B; 50 μm in C,D.

• Supplemental Figure S6 -

  Fig. S6. Confirmation of Notch1 deficiency in Igf1-rescued hair follicles. Immunohistochemical analyses with full-length Notch1 (1:50, rabbit, Abcam ab27526) and cleaved Notch1 (V1744, 1:100, rabbit, Cell Signaling, #2421) antibodies. Note the absence of Notch1 and V1744 immunoreactivity in emN1 and emN1;Ivl-IGF1 follicles. Scale bars: 50 μm.