Incubation system for microscopes

HeLa cells plated on 15 mm coverslips and grown in 12-well plates were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes at room temperature. The coverslips were washed in PBS, blocked with 5% bovine serum albumin (BSA) in PBS with 0.4% Triton X-100, then incubated with the indicated primary antibodies for 1 hour at room temperature or overnight at 4 °C. Following PBS wash, samples were incubated with secondary antibodies (Alexa Fluor 594 anti-mouse IgG [1:500], Alexa Fluor 488 anti-rabbit IgG [1:500]) for 30 minutes at room temperature in blocking solution. Cells were imaged using a fluorescence microscope (BIOREVO BZ-9000, Keyence). The images were quantified using GraphPad Prism (GraphPad software) and Image J software. Time-laps imaging was performed using fluorescence microscope (BIOREVO BZ-9000, Keyence) and Incubation System for Microscopes (TOKAI HIT).

U2OS cells were cultured in a 35-mm cell culture dish (Corning). Cells were co-transfected with non-silencing siRNA and GFP-tagged PALMD or siPALMD-1 and GFP vector. In the following day, cells were treated with 0.2 μg/ml ADR and the nucleus was visualized with Hoechst (Bisbenzimide H33342; Sigma, Kanagawa, Japan). Then, cells were incubated in an Incubation system for microscopes (TOKAI HIT, Shizuoka, Japan) under 5% CO₂ at 37 °C combined with a fluorescence microscope (Bio-Zero BZ-8000; Keyence, Osaka, Japan) equipped with a phase-difference lens (Nikon, Tokyo, Japan). The cells were captured at 5-min intervals. For the flourescence imaging, a halogen lamp was used with excitation (BP520-540HQ) and emission (BP555-600HQ) filters. Image acquisition and analysis were performed using analyzer software (BZ-H1TL; Keyence).