25 cm2 culture flasks

From P1 forward, keratinocytes were plated in triplicate to reduce handling-related variance. Keratinocytes were plated in 25 cm² culture flasks (Thermo Fisher Scientific) and passaged with a trypsin/EDTA solution generally when cultures reached 70–95% confluence or when more than seven days had gone by since seeding (see Supplementary Materials Table S1). Cultures were not allowed to reach confluence. Trypsin activity was inhibited by doubling the volume of the suspension with cAMP inducer-free ckDME-Ham. Cells were then centrifuged and resuspended in either ISO- or CT-supplemented ckDME-Ham. Seeding density for P1 to P3 ranged between 1 × 10⁵ cells/25 cm² and 3 × 10⁵ cells/25 cm² (see Supplementary Materials Table S1). Keratinocytes were maintained in culture and passaged until P3.

The HepG2 cell line was supplied by the Cell Culture Resource Centre at the University of Granada (Spain). Cells were precultured in 25 cm² culture flasks (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C using RPMI 1640 medium (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) fetal bovine serum and 2 mM glutamine in a humidified atmosphere of 5% CO₂. The culture medium for HepG2 was replaced once per day. After the cell density reached approximately 1 × 10⁶ cell/mL, cells were detached with trypsinization using a 0.25% trypsin-EDTA solution (Sigma Aldrich, St. Louis, MO, USA) and collected using centrifugation at 1500 rpm for 5 min. Cell density was determined using a Neubauer chamber.

Samples of human subcutaneous adipose tissue were obtained from patients who underwent surgery in the Department of Pediatric Surgery at Nihon University Itabashi Hospital (Tokyo, Japan). The patients gave their written informed consent, and the Ethics Committee of Nihon University School of Medicine approved this study. We prepared dedifferentiated fat (DFAT) cells using the ceiling culture method, as described previously [50 (link)]. Briefly, the adipose tissue (approximately 1 g) was cut into small pieces and digested with a 0.1% type I collagenase solution (Koken Co., Ltd., Tokyo, Japan). Cell samples were filtered and centrifuged at 135× g for 3 min, followed by collection of the floating cell layer containing mature adipocytes. The isolated adipocytes were washed with phosphate-buffered saline (PBS) and plated in 25 cm² culture flasks (Thermo Fisher Scientific) filled completely with CSTI-303MSC (Cell Science and Technology Institute, Miyagi, Japan) containing 20% FBS with 5 × 10⁴ cells per flask. The cells that adhered to the upper surface of the flasks were cultured for conversion to dedifferentiated fat cells. Fibroblast-like adhered cells (dedifferentiated fat cells) were cultured in 5 mL of CSTI-303MSC containing 20% FBS. Cells at passages 2–4 were used for differentiation experiments.

The HSF cells were cultured as monolayers in 25 cm² culture flasks (Nunc. Roskilde, Denmark) in RPMI 1640 medium supplemented with 10% FBS (v/v) and antibiotics at 37°C in a humidified atmosphere with 5% CO₂. The total number of cells used in the experiments was counted in a hemocytometer. 100 μl of cell suspension (1 × 10⁵ cells/ml) was added to appropriate wells of 96-well flat-bottomed microtiter plates. After 24 h of incubation, the medium was discarded and new media containing 2% FBS and appropriate concentrations of the AAF (12.5–200 μg mL^-1 range) were added. The HSF cells suspended in 100 μl of culture medium with 2% FBS without AAF addition were used as a control. The total number of cells was equivalent to that in the sample wells.

Samples were inoculated on solid medium (5 or 10 g/L agar) Z8 and Z8-salts (Rippka 1988 (link)). Isolations were carried out by repeated transfers (at least three times) of single cells or filaments on solid or liquid media under an inverted microscope (Nikon ECLIPSE TS100). Viable clones were then cultured in 25 cm² culture flasks (Nunc, Roskilde, Denmark) containing 10 mL of Z8. Cyanobacterial strains were maintained in the Paris Museum Collection (PMC) at 25°C, using daylight fluorescent tubes providing an irradiance of 12 μmol photons/m²/s, with a photoperiod of 16 h light/8 h dark. Isolated strains and cultures were all monoclonal and non-axenic.