Supplementary Figure 4: Functional expression of human PKD1 and mouse TRPP2 in Drosophila S2 cells.

(a) WNT9B binds to the surface of Drosophila S2 cells transfected with human PKD1 and mouse TRPP2. Confocal z-sections of non-permeabilized, untrasfected S2 cells incubated with 1 μg ml⁻¹ of purified WNT9B, PKD1/TRPP2-transfected but not incubated with purified WNT9B, or PKD1/TRPP2-transfected incubated with 1 μg ml⁻¹ of WNT9B, labeled with goat α-WNT9B detected with donkey α-goat coupled to Alexa-488. Insets show additional images of WNT9B-labeled cells from other fields. Scale bar, 5 μm (b) Expression levels of PKD1, TRPP2, or CD8α in transiently transfected S2 cells 48 h after the addition of CuSO₄ (700 μM) in the cultures to induce expression of PKD1, TRPP2 and CD8α. Experiments were successfully repeated three times (a and b). (c) Time course, step currents and I–V curves of WNT9B-induced whole cell currents in untrasfected (n = 9 cells pooled from 4 independent experiments) or PKD1/TRPP2-transfected S2 (n = 10 cells experiments) cells 48 h after the addition of CuSO₄. Step-currents represent steady-state currents at 5 min time point just before the addition of La³⁺. I–V curves were taken before the addition of 500 ng ml⁻¹ WNT9B (black lines) and 4 min after the addition of WNT9B just before the addition of La³⁺ (red lines). Statistical tests were performed using paired Student’s t-test, ^∗P < 0.05, ^∗∗P < 0.01. Data are shown as mean ± s.e.m.