Fig. 3.

Forced overexpression of mtTXNRD3 led to sorafenib resistance and a metabolic shift toward low OXPHOS and high glycolysis. (A) Forced overexpression of mtTXNRD3 in liver cancer cell line (SK-Hep1) by stable transfection. Immunoblot analysis was performed to detect the TXNRD3 protein expression in SK-Hep1 cells (OE, over expression; VC, vector control). “Mito” and “Cyto” indicate mitochondrial and cytosolic, respectively. (B) Relative levels (% of control) of the TrxR enzyme activity in SK-Hep1^OE cells compared with the SK-Hep1^VC cells. (C) Proliferation in SK-Hep1^OE and SK-Hep1^VC cells. Equal number (3 × 10³) of the indicated cells were started in cell culture, and MTS analysis was used to monitor cell growth every 24 h for up to 96 h. (D) Comparison of sensitivity of SK-Hep1^OE and SK-Hep1^VC cells to sorafenib. The indicated cells were treated with various concentrations of sorafenib for 72 h and cell viability was measured by MTS assay. (E, F) Effect of sorafenib on clonogenic formation in SK-Hep1 cells with or without overexpression of TXNRD3. Cells were treated with sorafenib as indicated. After two weeks of incubation, colonies were stained and imaged using an inverted microscope with a camera, and colony numbers were then counted. (G-J) Impact of mtTXNRD3 expression on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). OCR (G and I) and ECAR (H and J) of the indicated cells were monitored using the Seahorse XF24 analyzer. Data represent mean ± SD; **, p < 0.01; n = 3.