Fig. 4: Biopterin and CoQ reduction pathways are required for short-term cold resistance under Gpx4 dysfunction in hamster cells.

a Assessment of knock-out efficiency of ferroptosis-suppressors in HapT1 cells. Immunoblots of each protein in non-treated HapT1 (WT) or HapT1 cell populations, in which lentivirus vectors expressing SpCas9 and sgRNA (non-targeting control; sgNT, or sgRNA targeting each of three genes) were infected. b–d The proportion of dead cells during cold culture (4 °C) in the each HapT1 cell population infected with lentivirus vectors expressing SpCas9 and sgNT or sgRNA for FSP1 (b) or Dhodh (c) or Gch1 (d) in the presence or absence of 2 µM RSL3 (One-way ANOVA with the Tukey’s multiple comparison test, p < 0.05). e Biopterin synthesis and related pathways. f (upper panel) Experimental time course and (lower panel) the proportion of dead cells during cold culture in WT or Gch1 KO HapT1 cells, which were infected with lentivirus vectors expressing SpCas9 and sgNT or sgRNA for Gch1, in the presence of 6 µM ML210 with or without 100 µM BH₂ and 4 µM methotrexate (MTX). g Immunoblot of parental HT1080 and HT1080 cell populations in which FSP1 or Gch1 were exogenously overexpressed at 37 °C by lentivirus vector infection. h The proportion of dead cells during cold culture in parental HT1080 and HT1080 cell populations in which FSP1 or Gch1 were exogenously overexpressed (One-way ANOVA with the Dunnett’s multiple comparison test, ***p < 0.001).