Lcv110

We seeded hiPSCs in 0.5 µg/cm² laminin-511 E8-coated 35 mm dish (3000–035, ASAHI GLASS) with AK02N medium. After 5 days, iPSC colonies were formed. hiPSCs were exposed to normal medium or medium supplemented with 1.2 mol/L L-alanine for 2 h. Following incubation, the medium was replaced with normal medium containing 10 µg/mL PI (347–07881, Dojindo) and the cells were observed under a fluorescence microscope (LCV 110, Olympus, Tokyo).

For the random cell migration assay, cells were seeded onto a laminin-coated (10 μg/ml) glass-bottom dish and recorded under an inverted microscope system equipped with an incubator (LCV110; Olympus). For the neurite outgrowth assay (Fig S7B), the underside of 3-μm pore transwell membranes (Corning) was coated with 500 μl of 10 μg/ml laminin in PBS into a well of a 24-well plate. After coating, the membranes were removed from the laminin and placed into the well of a 24-well dish containing 500 μl differentiation media. A total of 100 μl of cell suspension (containing 1–2 × 10⁵ cells) was added to the insert chamber on top of the membrane. The cells were allowed to extend neurites through the membrane pores to the lower chamber (underside of the membrane) for 6 h at 37°C. The cells were then fixed and stained with an anti–α-tubulin antibody. Images were processed and analyzed using Fiji software (National Institutes of Health).

HeLa/Fucci2 cells were grown on 35 mm glass-bottom dishes in phenol red-free DMEM containing 10% FBS and transfected with either pME18Neo/Flag-Vpr-IRES-ECFP or the control pME18Neo/Flag-IRES-ECFP. The cells underwent long-term, time-lapse imaging using a computer-assisted fluorescence microscope (Olympus, LCV110) equipped with an objective lens (Olympus, UAPO 40X/340 N.A. = 0.90), a halogen lamp, a red LED (620 nm), a CCD camera (Olympus, DP30), and interference filters. For fluorescence imaging of FRET, we used the halogen lamp with a BP425-445HQ excitation filter (Olympus), a DM450 dichroic mirror (Olympus), and an FF01-542/27-25 emission filter (Semrock) for observing SCAT3.1. To quantify the results, the images of ECFP and Venus fluorescence were processed with MetaMorph 7.7.4 software (Universal Imaging), and the Venus/ECFP emission ratio was calculated.

The 2.0 × 10⁵ cells (200 μl suspension) were seeded onto the glass part (φ12 mm) of a glass base 35 mm dish (IWAKI). After 2 h incubation, the medium was replaced with the 2 ml fresh medium containing 20 mM HEPES and 100 μM D-luciferin, and the luminescence was imaged at each time point with a customized incubator microscope LCV110 (Olympus).

Raji cells were transiently transfected with 2 μg of an expression plasmid encoding GFP-tagged TAGLN2 (OriGene) and LifeAct-TagRFP (ibidi, Martinsried, Germany) using the Amaxa Cell Line Nucleofector Kit V (Lonza). For the preparation of cell images, cells 24 h after transfection were placed on a 35-mm glass dish with medium containing liquid collagen (Cellmatrix type 1-A, Nitta Gelatin Inc.), RPMI 1640, HEPES, NaHCO3, and NaOH (5 x 10^6 cells/ mL). In these experiments, the cells were stimulated with 10 μg/ mL F(ab’) anti-human IgM + IgG (eBioscience). Imaging data were acquired using an Incubator Fluorescence Microscope equipped with a multi-photon laser scanning system (LCV110 and FV1200MPE, Olympus) with a 25 x 1.05 NA water-immersion objective (XLPLNWMP, Olympus) and FV-10ASW software (Olympus). Fluorescence excitation was performed with a pulsed laser (InSight™ DeepSee™, Spectra Physics). The cells were kept at 37°C in 5% CO2 during the experiments. XYZT scanning data were acquired at 1.0-μm intervals in the Z-dimension and by Free Run in the T-dimension. Figures were processed using an open source Java image processing program, ImageJ (https://imagej.net/Welcome).

Time-lapse imaging was performed using an incubator microscope system (LCV110; Olympus Corp., Tokyo, Japan). Vinblastine sulfate (30 μM; Calbiochem, La Jolla, CA, USA) was added to the cells in which the Cellular Lights reagents had been introduced. The glass-based dishes were set up in a tray for the LCV110 system and preincubated for 60 min. Image acquisition was begun using a time interval of 15–20 min between each acquisition. These images were processed using the MetaMorph software (Molecular Devices, Inc., Sunnyvale, CA, USA).

AGS-EGFP/LMP1 or AGS-EGFP cells were mixed with AGS cells at a ratio of 2:98 and seeded on 35-mm glass-bottom culture dishes (μ-Dish; Ibidi). Mixed cells were incubated for 12–24 h until a monolayer formed. For time-lapse imaging, cells were monitored using the LCV110 (Olympus) incubator fluorescence microscope. Images were captured every 5 min and analyzed using Metamorph digital analysis software (Universal Imaging).