Quantifying Cytoskeletal Changes in Adipogenesis

Cells were cultured on disks at specific time points. Media was aspirated, and disks were washed in PBS. Cells were fixed with 4% PFA and permeabilized, incubated in blocking buffer, and then antibodies against adiponectin (Abcam, ab22554), vinculin (Abcam, ab18058), or YAP (SCBT, sc101199) overnight. Disks were washed, incubated in the appropriate second antibody for one hour, and mounted on glass slides for microcopy using a fluorescent microscope. Immunofluorescence images for adipogenesis and osteogenesis were quantified using MetaMorph. Vinculin and YAP were imaged with a fluorescent microscope and confocal microscope (Leica). YAP was manually quantified as predominantly nuclear versus cytoplasmic^{33 (link)}.