Active PKD1 inhibits breast tumour cell invasion. (a) MDA-MB-231 cells were transiently transfected with wildtype protein kinase D (PKD) 1, kinase-dead PKD1 (PKD1inactive) or constitutively-active PKD1 (PKD1active). After 24 hours cells were seeded on Matrigel-coated Transwell filters and Transwell invasion assays were performed over a time period of 16 hours. (b) Inducible expression of active PKD1 in MDA-MB-231-TR-PKD1active (PKD1.Y463E mutant) cells. Cells were treated with doxycyclin (DOX) for 16 hours. Cells were lysed and lysates were analysed by western blotting for expression of constitutively-active PKD1 (anti-FLAG) or actin (loading control). (c) MDA-MB-231-TR-PKD1.Y463E cells were seeded in 3D culture and were either left untreated for 12 (c1), 18 (c2) and 24 days (c4), or were treated with doxycyclin after 12 days of normal growth to induce the expression of active PKD1 (c3 and c5). Cells were photographed at day 12 (c1), day 18 (c2 and c3) or day 24 (c4 and c5). Arrows in c2 indicate cells invading from the spheroid into the extracellular matrix. Bars indicate the size of the spheroids.