Fig. 2: Serum protein APOH inhibits ferroptosis.

A Scheme for the procedure of MEFs cultured in Scm changed to medium containing 5% S-FBS adding 5% S-FBS or 5% R-FBS or 50–90% ASP or not for 24 h. The protein amount of 50–90% ASP was equal to 5% R-FBS. MEFs were treated with ERS2 in Scm for 24 h before cell viability was tested with CCK-8. ASP, ammonium sulfate precipitation. B Cell viability of MEFs cultured in indicated medium for 24 h and treated with ERS2 (0.1 μM) for 24 h in Scm according to the procedure shown in (A). C Cell viability of MEFs cultured in 5% or 10% of Rcm or Scm, or medium containing 5% S-FBS with the addition of various components, including 5% R-FBS, 5% Rmix, Q100–200, Q300–500, or SP100–500 for 24 h. Subsequently, the cells were treated with ERS2 (0.15 μM) for an additional 24 h in Scm. The protein amount of Rmix, Q100–200, Q300–500, and SP100–500 equals 5% R-FBS. Q100–200 or Q300–500, protein eluted with 100–200 mM or 300–500 mM NaCl in HiTrap-Q (Q) column; SP100–500, protein eluted with 100–500 mM NaCl in HiTrap-SP (SP) column. Rmix, the mixture of 60–80% ASP of R-FBS. D Venn diagram illustrates the overlapping proteins detected in the SP100–500 and Q300–500 fractions identified by mass spectrometry (MS). E Protein list of overlap proteins of SP100-500 and Q300–500 identified by MS and ranged by the intensity ratio of SP100–500 to Q300–500. F Cell viability of MEFs cultured in Scm changed for medium containing 5% S-FBS adding 5% R-FBS or different amounts of purified APOH for 24 h and treated with ERS2 (0.125 μM) for 24 h in Scm. Data are presented as mean ± s.d., n = 3 independent repeats. Unpaired, two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS not significant.