Fig. 3. WNT16B but not WNT16A enhanced keratinocyte cell proliferation. Growth curves of primary NHEKs retrovirally transduced with EGFP, WNT16A, WNT16B, -catenin or S33Y (constitutively active -catenin) determined by measuring total protein concentration of adherent cells (A) or ATP concentration (B) at the indicated time points. Insets show the relative growth rate at 50% confluency of each curve. (C) Cell-cycle profile determined by FACS (propidium iodide) analysis at the end of the cell proliferation assay (day 9) in A and B, with corresponding phase-contrast micrographs. (D) Primary NHEK transduced with either EGFP, WNT16A or WNT16B were cultured in low Ca²⁺ (0.06 mM) medium without irradiated 3T3 feeders and subcultured regularly up to passage 3. Phase-contrast and fluorescence micrographs were taken at post-transduction passage 1 and 3. (E) RTS3b keratinocytes were cultured at high cell density (2-3x10⁶ cells/35 mm²) for 7 days to induce contact inhibition and stratification-induced cell death. The number of viable adherent cells was quantified using CellTitre-Glo to measure the ATP concentrations of surviving cells at day 1 and 7. Each bar represents a mean ± s.e.m. of triplicate samples. ^*P<0.01 and ^***P<0.001 indicates statistically significant reduction in cell viability at day 7 compared with day 1.