pcdna3 2wnt16 v5  (Addgene inc)

Journal: bioRxiv

Article Title: Epidermal YAP Activity Drives Canonical WNT16/β-catenin Signaling to Promote Keratinocyte Proliferation in vitro and in the Murine Skin

doi: 10.1101/278705

Figure Lengend Snippet: Quantitative RT-PCR analyses showing WNT3, WNT3A, WNT4, WNT6, WNT7A, WNT7B, WNT10A, WNT10B and WNT16 expression levels relative to 18S or GAPDH in lysates of YAP2-5SA-ΔC skin (a) and HaCaT keratinocytes transfected with YAPS127A (b) or si YAP (c). Immunofluorescence staining of dorsal skin sections of adult wildtype and YAP2-5SA-ΔC littermate mice detecting K14 (e), YAP (f), active β-catenin (g), WNT16 (h), phospho-GSK3β (i). DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μ m.

Article Snippet: WNT16 overexpression was carried out using pcDNA3.2-WNT16-V5 (Addgene, 35942) and pcDNA3.2 vector control.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Epidermal YAP Activity Drives Canonical WNT16/β-catenin Signaling to Promote Keratinocyte Proliferation in vitro and in the Murine Skin

doi: 10.1101/278705

Figure Lengend Snippet: (a-d) Immunofluorescence staining of proliferating (top) and quiescent (bottom) HaCaT keratinocytes showing Ki67 (a), YAP (b), active β-catenin (c) and WNT16 expression (d). Western blots of (e) YAP, WNT16, GAPDH, and (f) phospho-GSK3β, total GSK3β, active β-catenin, total β-catenin, and GAPDH, in protein lysates of vector and YAPS127A transfected HaCaT keratinocytes ( N =3). Quantification of WNT16 expression (e.1), GSK3β phosphorylation (f.1), and active β-catenin expression (f.2).

Article Snippet: WNT16 overexpression was carried out using pcDNA3.2-WNT16-V5 (Addgene, 35942) and pcDNA3.2 vector control.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: Epidermal YAP Activity Drives Canonical WNT16/β-catenin Signaling to Promote Keratinocyte Proliferation in vitro and in the Murine Skin

doi: 10.1101/278705

Figure Lengend Snippet: (a) Fold-change of WNT16, CCND1 and cMYC expression normalized to GAPDH in HaCaT keratinocytes transfected with V5-WNT16 or vector control. (b) Western blots detecting V5-WNT16, active β-catenin, total β-catenin, and GAPDH in protein lysates of vector control and V5-WNT16 (right) transfected HaCaT keratinocytes ( N =3). (b.1) Quantification of fold-change protein expression of active β-catenin normalized to total β-catenin lysates of vector control and V5-WNT16 transfected HaCaT keratinocytes. (c) MTT proliferation assay using HaCaT keratinocytes transfected with vector control and V5-WNT16 ( N =3). Fold-change of WNT16 mRNA expression normalized to GAPDH in HaCaT keratinocytes transfected with esi GFP and esi WNT16 RNA (d). Western blots showing phospho-GSK3β expression normalized to total GSK3β (e and e.1) and active β-catenin normalized to total β-catenin (e and e.2) in protein lysates of esi GFP - and esi WNT16 -transfected (right) HaCaT keratinocytes. (f) MTT proliferation assay using HaCaT keratinocytes transfected with esi GFP and esi WNT16 RNA ( N =3).

Article Snippet: WNT16 overexpression was carried out using pcDNA3.2-WNT16-V5 (Addgene, 35942) and pcDNA3.2 vector control.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Proliferation Assay

Journal: bioRxiv

Article Title: Epidermal YAP Activity Drives Canonical WNT16/β-catenin Signaling to Promote Keratinocyte Proliferation in vitro and in the Murine Skin

doi: 10.1101/278705

Figure Lengend Snippet: (a) Working model of how YAP activity in keratinocytes promotes WNT16 production and canonical WNT/β-catenin signalling to drive cell proliferation in the murine epidermis. (b) Excessive WNT16 signals drive keratinocyte proliferation independently of β-catenin activity.

Article Snippet: WNT16 overexpression was carried out using pcDNA3.2-WNT16-V5 (Addgene, 35942) and pcDNA3.2 vector control.

Techniques: Activity Assay