Dapi nucleic acid binding dye

TED orbital fibroblasts were cultured in 24-well dishes until 90% confluent and then cultured in low serum DMEM (0.1%FBS) for 24 hours. PPIs (25 μM) or vehicle (DMSO) were then added to desired wells for 24 hours before treatment of some wells with 1 ng/mL TGFβ. PPIs were added to the wells every 24 hours for a total of 72 hours post TGFβ treatment. Cells were fixed with 4% paraformaldehyde for 20 minutes, then rinsed in phosphate-buffered saline and permeabilized with 0.3% Triton X-100. TED orbital fibroblasts were blocked with 3% bovine serum albumin, 1% rabbit serum, 3 mM glycine, 0.01% Triton X-100, and 0.1% Tween 20 in phosphate buffered saline for 1 hour. Cells were washed and incubated with Alexa-Fluor 488 conjugated mouse anti-αSMA antibody (Abcam, Cambridge, MA; cat. # ab202295) for 1 hour at room temperature (1:200). Afterwards, cells were washed three times in phosphate buffered saline and counter stained with DAPI nucleic acid binding dye (Invitrogen, Carlsbad, CA). The fluorescence was visualized on a ZOE fluorescent cell imager (Bio-Rad, Hercules, CA) utilizing the same settings for each image in the experiment.