Humidified chamber

Cells were cultured in a humidified chamber (Tokai Hit, Fujinomiya, Japan) maintained at 37°C under 10% CO₂. Time-lapse images were taken using an inverted microscope (BZ9000; Keyence, Osaka, Japan) with a 20x Plan Apo Fluor objective lens (Nikon, Tokyo, Japan) [27 (link), 28 (link)].

Fucci MPI-KO HT1080 cells (#3 subclone 9-5, 4 × 10⁴ cells/six-well dish) were cultured for 24 hr in 2.5 mL of complete medium supplemented with 20 μM Man. The cells were washed once with complete DMEM with no phenol red (040-30095, FUJIFILM Wako), 4 mM glutamine, and 10% FBS. The washed cells were cultured in 2.5 mL of the same medium supplemented with 50 μM Man or 5 mM Man in a humidified chamber (Tokai Hit) at 37°C with 5% CO₂. Fluorescence and differential interference contrast images were obtained every 15 min using KEYENCE BZ-X800 with a PlanFluor ×20 objective lens (NA = 0.45, WD = 8.80–7.50 mm, Ph1; KEYENCE) or EVIDENT FLUOVIEW FV10i. In KEYENCE BZ-X810, Fucci mCherry and mVenus signals were detected using a TRITC filter (Ex, 545/25 nm; Em, 605/70 nm; KEYENCE) and mVenus filter (Ex, 500/20; Em, 535/30 nm; M SQUARE), respectively. In EVIDENT FLUOVIEW FV10i, Fucci mCherry and mVenus signals were detected using mCherry (Ex, 559; Em, 570-620) and EYFP (Ex, 473; Em, 490-540) dye settings. Images were acquired after a 30 min equilibration period.

The cells were observed under a BZ-710X fluorescence microscope (Keyence, Osaka, Japan). The excitation and emission wavelengths were 360 and 460 nm for 4′,6-diamidino-2-phenylindole (DAPI), 490 and 516 nm for MTG, and 485 and 535 nm (green fluorescence) or 520–570 and 570–610 nm (red fluorescence) for JC-1, respectively. The absorption filters (Keyence) for DAPI (460 ± 50 nm), green fluorescent protein (525 ± 50 nm), and tetramethylrhodamine-5-(and -6-) isothiocyanate (605 ± 70 nm) were used for green, red and blue fluorescence, respectively. Phase contrast images were taken along with the fluorescence observations. During the observations, the cells were cultured in a humidified chamber (Tokai Hit, Shizuoka, Japan) set in the fluorescence microscope at 37 °C and 5% CO₂. A lens of 20× magnification for EdU, 10× magnification for SA-β-gal, or an oil-immersion objective lens of 60 × magnification for MTG and JC-1 was set approximately 30 min before the observations to keep the temperature of the lens constant. Observations were performed with a BZ-X Viewer microscope control application software (Keyence).