Priority Research Paper Volume 13, Issue 4 pp 4778—4793

A Wnt5a-Cdc42 axis controls aging and rejuvenation of hair-follicle stem cells

Inhibition of Cdc42 activity with CASIN induces a young like phenotype in old HFSCs and re-establishes canonical Wnt Signaling. (A) Cdc42 activity measured by a G-LISA in lysate of old Sca-1-/low keratinocyte cultured for 2 hours with the Cdc42 activity inhibitor CASIN, N=4 (B) Representative Cdc42 protein level in young, old and CASIN treated old Sca-1-/low keratinocytes by Western Blot (C) Densitometric score for the amount of Cdc42 protein in Sca-1-/low keratinocyte from young, old and old CASIN treated for 2 hours, N=3 (D) Distribution of Cdc42 (red) and tubulin (green) in young, old and old HFSCs cultured for 2 hours with CASIN by immunofluoresence, scale bar=5μm (E) Percentage of cells with a polar distribution of Cdc42 and tubulin in young, old and CASIN treated old HFSC cultured for 2hrs, N≥3, (F) Z-stacks and three-dimensional merged images of ß-catenin (red), nucleus (DAPI, blue) and their co-localization (yellow) in FACS sorted young, old and CASIN treated HFSC cultured for 2 hours by immunofluorescence (G) Quantification of the amount of nuclear ß-catenin in young, old and CASIN treated old HFSC cultured for 2 hours, N=3, (H) Level of expression of Wnt target genes in old HFSC cultured with CASIN for 6 hours, N=3, *P

Figure 3. Inhibition of Cdc42 activity with CASIN induces a young like phenotype in old HFSCs and re-establishes canonical Wnt Signaling. (A) Cdc42 activity measured by a G-LISA in lysate of old Sca-1-/low keratinocyte cultured for 2 hours with the Cdc42 activity inhibitor CASIN, N=4 (B) Representative Cdc42 protein level in young, old and CASIN treated old Sca-1-/low keratinocytes by Western Blot (C) Densitometric score for the amount of Cdc42 protein in Sca-1-/low keratinocyte from young, old and old CASIN treated for 2 hours, N=3 (D) Distribution of Cdc42 (red) and tubulin (green) in young, old and old HFSCs cultured for 2 hours with CASIN by immunofluoresence, scale bar=5μm (E) Percentage of cells with a polar distribution of Cdc42 and tubulin in young, old and CASIN treated old HFSC cultured for 2hrs, N≥3, (F) Z-stacks and three-dimensional merged images of ß-catenin (red), nucleus (DAPI, blue) and their co-localization (yellow) in FACS sorted young, old and CASIN treated HFSC cultured for 2 hours by immunofluorescence (G) Quantification of the amount of nuclear ß-catenin in young, old and CASIN treated old HFSC cultured for 2 hours, N=3, (H) Level of expression of Wnt target genes in old HFSC cultured with CASIN for 6 hours, N=3, *P<0.05, **P<0.01, ***P<0.001 (paired student’s t test). Error bars represent s.e.m.