Supplementary Information Targeted deletion of Crif1 in mouse epidermis impairs skin homeostasis and hair morphogenesis Jung-Min Shin 1 , Dae-Kyoung Choi 1 , Kyung-Cheol Sohn 1 , Ji-Young Kim 1 , Myung Im 1 , Young Lee 1 , Young-Joon Seo 1 , Minho Shong 2 , Jeung-Hoon Lee 1 , Chang Deok Kim 1 1 Department of Dermatology, Chungnam National University School of Medicine, Daejeon, Korea 2 Division of Endocrinology, Department of Internal Medicine, Research Center for Endocrine and Metabolic Diseases, Chungnam National University School of Medicine, Daejeon, Korea
P3 P5 K14-Cre; Crif1 fl/fl WT WT K14-Cre;Crif1fl/fl 1 2 1 2 1 2 3 1 2 3 Crif1 Crif1 Actin Actin Supplementary Figure 1. Western blot analysis of Crif1 in epidermal lysates from WT and Crif1 cKO mice at P3 and P5.
K14-Cre;Crif1 fl/fl WT esophagus small intestine large intestine Supplementary Figure 2. Histological examination of epithelial tissues in Crif1 cKO mice Sections of epithelial tissues from WT and Crif1 cKO mice at P5 were stained with hematoxylin and eosin (H&E), observing no differences between WT and Crif1 cKO mice. Scale bar, 100 μm
WT K14-Cre;Crif1 fl/+ (hetero) K14-Cre;Crif1 fl/fl (homo) P1 P3 P5 Supplementary Figure 3. Histological examination of WT, heteroxygous, and homo mice There was no histological difference between heteroxygous (K14-Cre; Crif1 fl/+ ) and WT (Crif1 fl/fl ) mice. Scale bar, 100 μm
K14-Cre;Crif1 fl/fl WT P1 P3 P5 Supplementary Figure 4. Immunohistochemical staining of mt-Co1 in epidermis of WT and Crif1 cKO mice. Scale bar, 200 μm .
P3 P5 K14-Cre;Crif1 fl/fl WT K14-Cre;Crif1 fl/fl WT K14 K1 Supplementary Figure 5. Immunofluorescence staining of K14 and K1 in epidermis of WT and Crif1 cKO mice. Scale bar, 100 μm
a b 4-OHT Crif1 fl/fl WT CreERT + + + + CreERT 4-OHT - + - + Crif1 Crif1 Actin Supplementary Figure 6. Establishment of Crif1 knockout model in vitro Primary keratinocytes cultured from Crif1 fl/fl mice were transduced with an adenovirus expressing CreERT (5 MOI) for 6 hrs. Cells were replenished, treated with 4-OHT and then cultured for a further 2 days. The expression of Crif1 was effectively reduced by 4-OHT treatment only in Crif1 fl/fl cells.
Toluidine blue staining (E18.5) WT K14-Cre;Crif1 fl/fl Supplementary Figure 7. Effect of Crif1 on skin barrier in embryonic development Toluidine blue staining was performed to examine the barrier defect in embryonic development. (embryonic day18.5) Crif1 cKO embryos showed normally developed epidermis.
K14-Cre;Crif1 fl/fl WT P3 P5 Supplementary Figure 8. TUNEL assay in epidermis of WT and Crif1 cKO mice. To examine the apoptotic cells, TUNEL assay was performed in epidermis of WT and Crif1 cKO mice at P3 and P5. Scale bar, 100 μm
K14-Cre;Crif1 fl/fl -1 K14-Cre;Crif1 fl/fl -2 K14-Cre;Crif1 fl/fl -1 K14-Cre;Crif1 fl/fl -2 WT-1 WT-2 WT-1 WT-2 75 100 Involucrin 63 75 48 63 Filaggrin 35 25 48 Actin 63 35 Loricrin 48 35 Supplementary Figure 9. Uncropped data for Figure 3b
Ad/CreERT Ad/CreERT - + 4-OHT - + 4-OHT 25 Crif1 20 Actin 48 35 35 Caspase3 25 Cleaved 20 Caspase3 17 11 Supplementary Figure 10. Uncropped data for Figure 4f
K14-Cre;Crif1 fl/fl -1 K14-Cre;Crif1 fl/fl -2 cytosolic nucleus cytosolic nucleus WT-1 WT-2 + + + + Ad/CreERT Ad/CreERT + + + + - + - + 4-OHT - + - + 4-OHT 100 b -catenin 25 Crif1 75 20 63 a -tubulin 48 Actin 48 35 100 b -catenin 75 75 LaminB 63 Supplementary Figure 11. Uncropped data for Figure 5a and d
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