Cyclooxygenase inhibition plays an important role in cardiomyocytes differentiation. TCF reporter assay using IMR90-WNT reporter line: (a,b) The effect of Sulindac and IWP2 was examined. On day 0, CHIR99021 (10 μM) was added to activate Wnt signaling and TCF promoter activity from day 2 to day 4 cells were treated with Sulindac and IWP2. Fluorescence was recorded and images were captured on day 4. Scale bar, 100 μm (a), Error bars, ±SD; n = 3 independent biological replicates, (Student’s t test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) (b); (c) Representative FACS analyses for percent eGFP+ cells generated using Nimesulide, Diclofenac and Piroxicam. Control (red) indicates undifferentiated HES3 cells. (d) Representative FACS analyses for percent cTnT+ cells generated using COX-1, COX-2 and COX-1+2 siRNA. Undifferentiated IMR90 cells were used as control. (e,f) IMR90-WNT reporter cells were transfected with COX-1, COX-2 and COX-1+2 siRNAs after treatment with CHIR99021. Fluorescence images were captured on day 4 and analysed using ImageJ to obtain relative GFP intensities. Scale bar = 50 μm, Error bars, ±SEM; n = 3 independent biological replicates, (Student’s t test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).