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Fig. 2. Expression and detection of WNT16A and WNT16B isoforms in cultured human keratinocytes. (A) Immunocytochemistry using WNT16 antibody on retrovirally transduced primary NHEKs expressing EGFP, WNT16A or WNT16B. (B) RT-PCR gel electrophoresis detection of WNT16A or WNT16B mRNA expression using isoform-specific primers in NHEKs and RTS3b keratinocytes transduced with either WNT16A (16A), WNT16B (16B) or EGFP (C). GAPDH was amplified as a loading control. WNT16A/B expression plasmids were used as positive controls for each isoform. (C) Western blot detection of WNT16 protein in NHEKs and RTS3b keratinocytes transduced with either WNT16A or WNT16B. A lower molecular mass product (30-35 kDa; X) was detected in WNT16A-tranduced cell lysates. Corresponding fluorescence micrographs show the expression level of WNT16A or B in respective samples before harvesting for immunoblotting. (D) In vitro protein translation of pcDNA-WNT16A or B expression plasmids produced expected size proteins (
40 kDa for both isoforms). pGL3 (luciferase gene) and pcDNA3.1 were used as positive (+ve) and negative (–ve) controls, respectively. (E) The in vitro translated protein lysates (from D) were subsequently used for immunoblotting with the WNT16 antibody, showing positive immunoreactivity in both WNT16A and B isoforms. (F-H) WNT16 isoforms do not activate through a canonical WNT signalling pathway in human keratinocytes. (F) Immunocytochemistry of endogenous 
-catenin proteins (using a pan--catenin antibody) in primary NHEKs transduced with either EGFP or WNT16B or control cells treated with LiCl (5 mM, 24 hours). (G) TCF/LEF luciferase reporter assay on primary NHEKs co-transduced with pSIN-OT and either EGFP or WNT16B. CTRL cells were mock transduced with retrovirus bearing no expression gene. Positive control cells (for activation of canonical WNT signalling) were treated with LiCl (5 mM) to activate LEF/TCF promoter activity. (H) TCF/LEF luciferase reporter assay on 293T cells co-transfected with EGFP, WNT1 or WNT16B. pOT (grey bar) or pOF (black bar) indicate cells co-expressing luciferase reporter bearing an active or mutant TCF/LEF binding sites, respectively. ***P<0.001 indicates significant activation of TCF/LEF promoter activity. (I) Immunoblotting of WNT16B, -catenin, phospho-Jun, phospho-JNK, and GAPDH in the indicated cell types expressing either WNT16B or EGFP.
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