Oil red o

DPSCs were differentiated into adipocytes, osteoblasts, or chondrocytes using adipogenic osteogenic or chondrogenic differentiation-inducing medium (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. Cells were stained with Oil red O (Polysciences, Warrington, PA) and fatty acid-binding protein-4 (FABP-4; R & D Systems, Minneapolis, MN) to assess adipogenic differentiation. Cells were stained with alkaline phosphatase (ALP; Millipore, Billerica, MA) and osteocalcin (R & D Systems) to assess osteogenic differentiation. Cells were stained with aggrecan (R & D Systems) to assess chondrogenic differentiation. For the detection of nuclei, cells were stained with 4′-6-Diamidino-2-phenylindole (DAPI; Sigma Aldrich, St. Louis, MO).

For the differentiation assay, fresh-DPSCs and cryo-DPSCs at passage 3 to 4 were cultured for 2–3 weeks in the induction medium according to the manufacturer’s instructions (R&D Systems). Adipogenic differentiation medium contained 1 % h-insulin, 2 % L-glutamine, 10 % mesenchymal cell growth supplement, 0.5 % dexamethasone, 0.2 % indomethacin, and 0.1 % 3-isobutyl-methyl-xanthine. Adipogenic maintenance medium contained 1 % h-insulin, 2 % L-glutamine, and 10 % mesenchymal cell growth supplement. For the detection of adipogenic differentiation, we stained the lipid accumulation with oil red O (Polyscience, Warrington, PA, USA).
Osteogenic induction medium contained 0.5 % dexamethasone, 2 % L-glutamine, 0.5 % ascorbate, 1 % β-glycerophosphate and 10 % mesenchymal cell growth supplement. We detected osteogenic differentiation with alkaline phosphatase (ALP) activity using an ALP detection kit (Millipore).

Larvae were fixed in 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA) overnight at 4°C, stained with oil red O, and scored for steatosis as previously described [35] (link). Cryosections were stained by immersing slides in increasing concentrations of propylene glycol (85%, 100%) for 10 minutes followed by an overnight incubation in oil red O (0.5% in propylene glycol, Polysciences, Warrington, PA). Excess oil red O was removed the next day by sequential washes in 100% and 85% propylene glycol for 5 minutes. Nuclei were counterstained with hematoxylin.
HepG2 and 293T cells were stained as previously described [57] (link) and counterstained with hematoxylin. oil red O droplet area and number were calculated using ImageJ. In brief, images were split into the red, green, and blue channels and the green channel was further processed for quantification as described, as oil red O has an excitation at 510 nm [58] (link). Following this, the background was subtracted from each image to ensure only counting of oil red O droplets. Droplets equal or greater than five square pixels were counted and area quantified. The values were copied into Microsoft Excel and the average droplet area and number of droplets per nucleus were counted for each field. Twenty independent fields from nATF6 and GFP-transfected HepG2 and 293T cells were imaged at 60x magnification.