G3250

To determine induction of apoptosis, a commercially available kit from Promega (G3250; Madison, WI) to measure fragmented DNA by incorporating fluorescein 12-dUTP at 3′-OH DNA ends using the Terminal Deoxynucleotidyl Transferase enzyme was used following manufacturer’s protocol. Briefly, 500,000 cells were plated in a 6-well plate. Cells were allowed to adhere overnight then treated with 2.4 μg/mL microsclerodermin A (2X IC₅₀ for NFκB inhibition) or its vehicle control for 24 hours. At the end of treatment, cells were trypsinized, fixed with 4% paraformaldehyde and permeabilized with ice cold ethanol. Cells were transferred to a 96 well U-bottom plate, equilibrated for five minutes and then treated with 25 μl of a reaction mix containing the nucleotide mix and the enzyme to label the fragmented DNA for 60 minutes at 37°C. The reaction was stopped by the addition of 20mM EDTA. The resulting fluorescence was visualized via flow cytometry in a BD FACSCanto equipped with a plate adapter, acquiring 20,000 events. Statistical significance was determined using a student’s T test comparing results to vehicle control.

We dissected TMJs from β-catenin(ex3)^Co12ER mice and Cre-negative control mice. Samples were fixed in 10 % formalin, decalcified, and embedded in paraffin. The condyles were sectioned into serial sections at 3 µm thick in an anterior-posterior direction. Cell proliferation was carried out using anti-PCNA antibody at the dilution of 1:200 (abl8197, ABCAM®, Cambridge, UK). Apoptosis assay was carried out using a TUNEL assay kit according to the manufacturer’s instructions (G3250, Promega, Madison, WI, USA).