Fig. 4. Silencing endogenous WNT16B gene expression decreased keratinocyte proliferation and cell survival. (A) Fluorescence micrographs showing isoform-specific siRNA gene silencing in WNT16A or WNT16B-transduced RTS3b keratinocytes transfected with either shCtrl (control shRNA with random sequence), shA (against WNT16A), shB (against WNT16B) or shAB (against both WNT16 isoforms). All cells were maintained in culture medium containing puromycin (2 µg/ml) to select for shRNA-expressing cells. (B) Pixel densitometry of fluorescence images in A. Each bar represents % mean ± s.e.m. (n=5 images) of green fluorescence. ^***P<0.001 indicates statistically significant gene knockdown by respective shRNA expression. (C) Western immunoblotting of WNT16B protein (arrows) from cell lysates collected from WNT16-expressing NHEK or RTS3b cells transfected with each shRNA, as indicated, confirming the specific knockdown of WNT16 protein expression by RNAi. (D) RTS3b keratinocytes transduced with either EGFP or WNT16B or mock transduced (empty virus, grey bar) were transfected with each of the indicated shRNAs and cultured at high density for 7 days to promote stratification-induced cell death. Cells transfected with shRNA were grown in culture medium containing puromycin (2 µg/ml) to select for shRNA-expressing cells. Viable cells were quantified at post shRNA transfection day 1 and 7. Each bar represents mean ± s.e.m. of three samples. ^*P<0.05 and ^**P<0.01 indicate statistically significant effects induced by shRNA expression. This experiment has been reproduced twice on separate occasions with similar results. (E,F) Silencing -catenin attenuated 293T cell proliferation. (E) Transient co-transfection of siCat (10 nM, 48 hours) with EGFP, Wnt1 or -catenin in the presence of pGL3-OT luciferase reporter in 293T cells. Each bar represents a mean ± s.e.m. of triplicate samples. ^**P<0.01 and ^***P<0.001 indicate statistically significant inhibition of TCF/LEF promoter activity by siCat. (F) Transient co-transfection of siCat in the absence (CTRL) or presence of either EGFP or WNT16B expression vectors in 293T cells. Cell density at day 7 post-transfection was determined using CellTitre-Glo to measure ATP concentrations of surviving cells. Each bar represents mean ± s.e.m. n=6. ^*P<0.05 and ^**P<0.01 indicate statistically significant inhibition of cell proliferation by siCat. (G,H) Constitutive expression of WNT16B induces hyperproliferation in organotypical culture system. (G) Representative haematoxylin and eosin sections of organotypic cultures using EGFP, WNT16A or B-transduced NHEK or RTS3b cells seeded on de-epidermalised dermis (DED). (H) Area quantification of epidermal thickness in organotypical cultures performed in A. Each bar represents a mean epidermal area above the DED of four independent organotypical cultures performed on separate occasions. ^**P<0.01 and ^***P<0.001 indicate statistically significant thickening of epidermal layer above the DED.