Extended Data Fig. 4: Expression pattern and subcellular localization of GPX4 isoforms.
From: DHODH inhibitors sensitize to ferroptosis by FSP1 inhibition
a. Structural organization of the GPX4 gene, mRNA and protein of the GPX4 isoforms. Arrows indicate the transcription initiation sites. The dashed lines indicate the different splicing variants. ATG indicates the initiation codon of methionine. MTS, mitochondrial targeting sequence; NLS, nuclear localization signal. NLS also functions for stretching of DNA binding motifs. b. A scheme depicting the reported subcellular localization of each GPX4 isoform in somatic and testicular cells. The short form is abundantly expressed in the cytoplasm and mitochondrial extra-matrix space of somatic cells, while the mitochondrial matrix form is abundantly expressed in the mitochondrial matrix of testicular cells. The illustration was created using BioRender.com (a, b). c. Viability of GPX4 KO HT-1080 cells (500 cells/well) overexpressing the short or mitochondrial matrix form of GPX4 for three days after withdrawal of ferrostatin-1 (a ferroptosis inhibitor). The cells were prepared by infection with the indicated serial dilution of lentiviral particles containing the expression plasmids. Immunoblotting validated the overexpression of each form. Viability of the cells incubated with Lip1 (1 μM) was taken as 100%. d. The design of the primer pairs detecting both the short and mitochondrial matrix forms (106 bp) and specific for the mitochondrial matrix form (196 bp). Agarose gel images show the amplification of the specific single band. The ratio of the mitochondrial matrix form/short and mitochondrial matrix forms of GPX4 mRNA expression in the cancer cell lines was calculated as 2−ΔCT in quantitative RT-PCR. Data is representative of two independent experiments (c and d). Data is mean ± s.d. of n = 3 (c and d).
