Figure 1

RITA treatment promotes the formation of covalently linked TrxR1 oligomers in cancer cells that can be prevented by NDGA and correlate with cell death. (a) RITA (1 μM) treatment of HCT116 cells for 8 h triggers the formation of an ≈110 kDa TrxR1-positive band seen in western blots.¹³ Here TrxR1-derived protein species were enriched from HCT116 cells (controls or treated with RITA, as indicated) by immunoprecipitation, visualized by Coomassie staining and further analyzed by tryptic digests and mass spectrometry (Supplementary Table S1). (b, c) Pre-treatment of HCT116 cells using 50 μM NDGA for 1 h blocked RITA-induced formation of TrxR1 dimeric bands as seen on western blots, also further verified using knockdown of TrxR1 with siRNA (Siseq1 and Siseq2). Below the western blots, the results of densitometric quantifications of dimer over monomer ratios are shown. See text for details. DEDA, 7,7-dimethyl‐(5Z,8Z)‐eicosadienoic acid 25 or 50 μM (phospholipase A2 inhibitor, sPLA2 and cPLA2); Indo, indomethacin 20 μM (Cox 1, 2 inhibitor); MAFP, methyl arachidonyl fluorophosphonate 10 μM (phospholipase A2 inhibitor, cPLA2 and iPLA2); NDGA, nordihydroguareric acid 50 μM; PM, pyridoxamine dihydrochloride 2 mM; SA, salicylamine 2 mM (to scavenge lipid‐derived ketoaldehydes). (d) NDGA treatment prevents RITA-induced cell death in HCT116 cells. (e) TrxR1 oligomers in cell lysates, as indicated, were fractionated using gel filtration (left), whereupon all fractions were analyzed for TrxR activity (middle) and band sizes as detected using western blot of reducing SDS-PAGE analyses (right). The expected migration over the gel filtration column of different TrxR1 oligomers in solution is also indicated as the number of TrxR1 subunits that would be required to yield the corresponding elution (top, right panel)