Cellular TrxR1 activity assay

After HCT-116 or SW620 cells were treated with different concentrations (2-fold dilution for 11 concentrations starting at 100 µM) of HT for 24 h, the cells were harvested and washed twice with PBS. Total cellular proteins were extracted using RIPA buffer for 30 min on ice and quantified using the BCA method. TrxR1 activity in cell lysates was measured via the endpoint insulin reduction assay. Briefly, the cell extract containing 20 µg total protein was incubated in a final reaction volume of 50 µl containing 100 mM Tris-HCl (pH 7.6), 0.3 mM insulin, 660 µM NADPH, 3 mM EDTA and 1.3 µM recombinant human Trx for 30 min at 37˚C. The reaction was terminated by adding 200 µl of 1 mM DTNB in 6 M guanidine hydrochloride, pH 8.0 at 25˚C for 5 min. A blank sample, containing everything except Trx, was treated in the same manner. The AB at 412 nm was measured, and the blank value was subtracted from the corresponding absorbance value of the sample. The same amount of DMSO was added to the control experiments and the TrxR1 inhibitory rate was calculated using the following formula: TrxR1 Inhibitory rate=[1-(AB value of sample-AB value of blank)/(AB value of control-AB value of blank)] x100%.