Figure 5.

mtp53 mediated ferroptosis contributes to low-concentration PTX and RSL3-induced synthetic cell death. (A) Western blotting analysis of p53 in Detroit562 treated with PTX at indicated low concentrations for 24 hrs. Protein bands were quantified with Image J. The quantification reflected the relative amounts as a ratio of each protein band relative to the lane’s loading control (GAPDH). (B) Detroit562 cells were transfected with pCDNA3-GFP-P53 (R175H) plasmid then fixed and stained with anti-P53(R175H) monoclonal antibody (Cat. # 26072) and with secondary antibody. Fluorescent images were collected simultaneously (Scale bar=25 µm). (C) Detroit562 and FaDu cells were transfected with p53 siRNA (sc-29435) or its relative negative control was treated with combination of RSL3(2μM) and low-concentration PTX (2 nM) for 24 hrs. Cell viability was assayed using Cell Counting Kit-8. (D) Detroit562 and FaDu cells were transfected with p53 siRNA (sc-29435) or its relative negative control was treated with RSL3 (2 µM) and low-concentration PTX (2nM) for 24 hrs with or without Lip-1 (0.02 µM) pretreatment. Lipid ROS level (Percentage of max) was determined using the C11-BODIPY, samples were examined using BD FACS Calibur. (E) Graphical representation of the histogram for relative lipid ROS. (F) Western blotting analysis of p53 and SLC7A11 in Detroit562 and FaDu cells transfected with p53 siRNA (sc-29435) or its relative negative control. (G, H) Western blotting analysis of SLC7A11 in Detroit562 and FaDu cells transfected with p53 siRNA (sc-29435) or its relative negative control. Detroit562 and FaDu cells were treated with low-concentration PTX (2 nM), RSL3 (2 µM), combination drug with or without the pretreatment of Lip-1 (0.02 µM) for 24 hrs. Data represent the means±SD, n=3 independent experiments. ns p>0.05, **p<0.01, ***p<0.001, ****p<0.0001, paired t-test.