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

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

Wnt5a regulates Cdc42 activity and induces an old like phenotype in young HFSCs. (A) Cdc42 activity measured using G-LISA kit in lysate of young Sca-1-/low keratinocyte after Wnt5a treatment for 2 hours, N=4 (B) Representative Western blot on total Cdc42 protein levels in Wnt5a treated young Sca-1-/low keratinocytes (C) densitometric score of western blot results for total Cdc42 in young Sca-1-/low keratinocyte after 2 hours of Wnt5a treatment N=3 (D) Cdc42 (red) and tubulin (green) distribution in young and old HFSC with or without Wnt5a treatment for 2 hours scale, immunofluorescence, scale bar=5μm (E) Percentage of cells polar for the distribution of Cdc42 and tubulin in young and old HFSC after Wnt5a treatment for 2 hours, 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 and old HFSC by immunofluoresence (G) Quantification of the ß-catenin signal in the nucleus and cytoplasm of young, old and Wnt5a treated HFSCs, N=3 (H) Expression levels of Axin-2 in young HFSC after Wnt5a treatment, N=3 (I) Morphology of colonies formed by young HFSCs after Wnt5a treatment (J) Number of colony forming units in young, aged and Wnt5a treated HFSCs, N=3 (K) Morphology of colonies formed by old Sca-1-/low keratinocytes transduced (green) with non-targeted scrambled (NT-GFP+) and targeted Wnt5a knock down (Wnt5aKD-GFP+) sh-RNA (L) Number of colony forming units in old Sca-1-/low keratinocyte transduced (green) with non-targeted scrambled (NT-GFP+) and targeted Wnt5a knock-down (Wnt5aKD-GFP+) sh-RNA, N=3, *P

Figure 2. Wnt5a regulates Cdc42 activity and induces an old like phenotype in young HFSCs. (A) Cdc42 activity measured using G-LISA kit in lysate of young Sca-1-/low keratinocyte after Wnt5a treatment for 2 hours, N=4 (B) Representative Western blot on total Cdc42 protein levels in Wnt5a treated young Sca-1-/low keratinocytes (C) densitometric score of western blot results for total Cdc42 in young Sca-1-/low keratinocyte after 2 hours of Wnt5a treatment N=3 (D) Cdc42 (red) and tubulin (green) distribution in young and old HFSC with or without Wnt5a treatment for 2 hours scale, immunofluorescence, scale bar=5μm (E) Percentage of cells polar for the distribution of Cdc42 and tubulin in young and old HFSC after Wnt5a treatment for 2 hours, 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 and old HFSC by immunofluoresence (G) Quantification of the ß-catenin signal in the nucleus and cytoplasm of young, old and Wnt5a treated HFSCs, N=3 (H) Expression levels of Axin-2 in young HFSC after Wnt5a treatment, N=3 (I) Morphology of colonies formed by young HFSCs after Wnt5a treatment (J) Number of colony forming units in young, aged and Wnt5a treated HFSCs, N=3 (K) Morphology of colonies formed by old Sca-1-/low keratinocytes transduced (green) with non-targeted scrambled (NT-GFP+) and targeted Wnt5a knock down (Wnt5aKD-GFP+) sh-RNA (L) Number of colony forming units in old Sca-1-/low keratinocyte transduced (green) with non-targeted scrambled (NT-GFP+) and targeted Wnt5a knock-down (Wnt5aKD-GFP+) sh-RNA, N=3, *P<0.05, **P<0.01, ***P<0.001 (paired student’s t test). Error bars represent s.e.m.