Fig. 4

RRAD enhances ferroptosis in pancreatic cancer cells. A, B The shRNAs against RRAD plasmid were transfected into SW1990 cell line. Plasmid overexpressing RRAD was transfected into Mia PaCa-2 cell line. Western blot analysis and qPCR analysis were performed to exam SETD8 protein and mRNA levels, respectively. C The MDA was detected in RRAD-silenced SW1990 cell line and RRAD-overexpressed Mia PaCa-2 cell line. The results showed that RRAD could promote the expression of MDA. D The GSH/GSSG ratio was detected in RRAD-silenced SW1990 cell line and RRAD-overexpressed Mia PaCa-2 cell line. It was found that silencing RRAD could increase the GSH/GSSG ratio and overexpression of RRAD could decrease the GSH/GSSG ratio. E Cell viability was detected in the RRAD-silenced SW1990 cell line in the presence or absence of 2 μmol/l RSL3 and the RRAD-overexpressed Mia PaCa-2 cell line in the presence or absence of 2 μmol/l Fer. The results showed that the cell viability increased by RRAD knockdown could be reversed by the ferroptosis inducer RSL3 and the cell viability decreased by RRAD overexpression could be reversed by the ferroptosis inhibitor Fer. F Flow cytometry analyzed the fluorescence of BODIPY581/591C11 (lower) and the relative content was calculated (upper). The results showed that RRAD could increase the level of intracellular lipid peroxidation. Two tailed unpaired Student t-test was used in the above experiments. *P < 0.05; **P < 0.01