Targeted deletion of Crif1 in mouse epidermis impairs skin - - PowerPoint PPT Presentation

Targeted deletion of Crif1 in mouse epidermis impairs skin homeostasis and hair morphogenesis

Supplementary Information 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

Crif1 Actin Crif1 Actin WT K14-Cre; Crif1fl/fl 1 2 1 2 WT K14-Cre;Crif1fl/fl 1 2 3 1 2 3 P3 P5 Supplementary Figure 1. Western blot analysis of Crif1 in epidermal lysates from WT and Crif1 cKO mice at P3 and P5.

WT K14-Cre;Crif1fl/fl 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

P1 P3 P5 WT K14-Cre;Crif1fl/+ (hetero) K14-Cre;Crif1fl/fl (homo) Supplementary Figure 3. Histological examination of WT, heteroxygous, and homo mice There was no histological difference between heteroxygous (K14-Cre; Crif1fl/+) and WT (Crif1fl/fl) mice. Scale bar, 100 μm

WT K14-Cre;Crif1fl/fl P1 P3 P5 Supplementary Figure 4. Immunohistochemical staining of mt-Co1 in epidermis of WT and Crif1 cKO mice. Scale bar, 200 μm.

WT K14-Cre;Crif1fl/fl WT K14-Cre;Crif1fl/fl P3 P5 K14 K1 Supplementary Figure 5. Immunofluorescence staining of K14 and K1 in epidermis of WT and Crif1 cKO mice. Scale bar, 100 μm

CreERT 4-OHT Crif1

a

CreERT 4-OHT WT Crif1fl/fl Crif1 Actin + + + +

• +
• +

b

Supplementary Figure 6. Establishment of Crif1 knockout model in vitro Primary keratinocytes cultured from Crif1fl/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 Crif1fl/fl cells.

WT K14-Cre;Crif1fl/fl Toluidine blue staining (E18.5) 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.

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 WT K14-Cre;Crif1fl/fl P3 P5

Involucrin

75 100 63

Loricrin

63 48 35

WT-1 WT-2 K14-Cre;Crif1fl/fl-1 K14-Cre;Crif1fl/fl-2

63 75 48 35 25

Filaggrin

48 35

Actin WT-1 WT-2 K14-Cre;Crif1fl/fl-1 K14-Cre;Crif1fl/fl-2 Supplementary Figure 9. Uncropped data for Figure 3b

25 20

Crif1 Caspase3 Cleaved Caspase3

25 35 20 17 11

Actin

48 35

• + 4-OHT - + 4-OHT

Ad/CreERT Ad/CreERT Supplementary Figure 10. Uncropped data for Figure 4f

Crif1

25 20

b-catenin

75 100

a-tubulin

63 48 75 63

LaminB

+ + + +

• +
• +

Ad/CreERT 4-OHT cytosolic nucleus

+ + + +

• +
• +

Ad/CreERT 4-OHT cytosolic nucleus

48 35

Actin b-catenin

100 75

WT-1 WT-2 K14-Cre;Crif1fl/fl-1 K14-Cre;Crif1fl/fl-2 Supplementary Figure 11. Uncropped data for Figure 5a and d