FIGURE 4.

TrxR is a target for santamarine in cells. (A) Santamarine inhibited TrxR activity in live HeLa cells, as assessed by TRFS-green imaging. The top panel (bright field pictures) and the bottom panel (fluorescence pictures) were acquired by an inverted fluorescence microscope. The images represent three independent experiments performed in triplicate. Scale bar: 20 μm. (B) The effect of santamarine treatment on TrxR activity of HeLa cells was evaluated by the endpoint insulin reduction assay. HeLa cells were treated with different concentrations of santamarine for 24 h, and then TrxR activity in cell lysate was measured using an endpoint insulin reduction assay and expressed as a percentage of the DMSO-treated control. The inset in (B) reflects the time-dependent inhibition of TrxR by santamarine in HeLa cells. After the HeLa cells were treated with 50 μM santamarine for an indicated time point, the TrxR activity in cell lysate was also measured by the endpoint insulin reduction assay. (C) and (D) TrxR is indeed involved in the cellular process of santamarine. (C) Knockdown of TrxR1 sensitizes the cells to santamarine treatment. After 48 h of treatment of HeLa-shNT cells (control) and HeLa-shTrxR1 cells (knockdown) with the indicated concentrations of santamarine, the cell viability was determined by MTT assay. (D) Overexpression of TrxR1 protects the cells from santamarine treatment. The HEK-IRES cells (control) and HEK-TrxR1 (overexpression) cells were with the designed concentrations of santamarine for 48 h, and the cell viability was assessed by the MTT assay. All data are from three independent experiments in triplicate. **p < 0.01 versus the control group in (B), between two cell lines in (C) and (D).