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. 2021 Jan 9;26(2):315. doi: 10.3390/molecules26020315

Figure 3.

Figure 3

LH exhibited effective inhibition on Trx/ TrxR system. In vitro assays for Trx1 (A), TrxR (B), Grx1 (C), GR (D). For Trx1 activity assay, 0.04 µM Trx1, 1 µM TrxR was incubated with LH. For TrxR activity assay, 0.4 nM of TrxR, 2 µM TrxR was incubated with LH. For Grx activity assay, 0.6 nM of hGrx-1, 50 nM GR, 0.5 mM GSH was incubated with LH. For GR activity assay, 20 µM of GR, 1 mM GSH was incubated with LH. (E), TrxR was pre-reduced with NADPH for 30 min with indicated concentrations of LH, followed by addition of DTNB. (F), 50 µM LH was pre-incubated or no pre-incubated with TrxR for 30 min, followed by addition of DTNB. (G), NADPH-reduced TrxR was incubated with 10 µM LH for 0, 5, 10, 20 min. (H), Expression levels of Trx1 and TrxR1 in SW1990 and PANC-1 cells were determined after incubating with indicated concentration of LH for 24 h by Western blotting. β-tublin served as protein-loading control. (I), Knockdown efficiency was analyzed after SW1990 cells transfected with TrxR siRNA, control siRNA and positive GAPDH siRNA for 48 h by Western blotting. (J), TrxR-silenced SW1990 cells and control cells were treated with 0, 2.5, 5, 8, 10, 20 µM LH for 48 h, and cell viability was measured by CCK8 assay. (K), SW1990 and PANC-1 cells were incubated with indicated concentration LH for 48 h. Total and activation forms of ASK1 and JNK were evaluated by Western blotting. GAPDH is shown as a protein-loading control.