Microdishes

LNCaP cells were plated at a density of 100 000 cells per plate on Ibidi microdishes (Ibidi, LLC, Verona, WI) and cultured at 37 °C in RPMI-1640 medium supplemented with 10% FBS and 2 mM L-glutamine. After 48 h in culture, cells were transfected with a GFP-tagged recombinant PKCε construct using X-tremeGENE HP DNA transfection reagent (Sigma) according to the manufacturer’s recommendations. After 24 h, the cells were treated as indicated with 1 μM of PMA and 3 μM of DAG–lactone derivatives in confocal medium (Dulbecco’s modified Eagle medium without phenol red supplemented with 1% FBS), and time-lapse images were collected every 30 s using the Zeiss AIM software. Imaging was with a Zeiss LSM 510 confocal microscopy system (Carl Zeiss, Inc.) with an Axiovert 100 M inverted microscope operating with a 25 mW argon laser tuned to 488 nm. A 63 × 1.4 NA Zeiss Plan-Apochromat oil-immersion objective was used together with varying zooms (1.4 to 2×). The imaging was performed using the resources of the Confocal Microscopy Core Facility, Center for Cancer Research, National Cancer Institute.