Fig. 5: The nucleotide A575 in GPX4 (NM_002085) transcript is critical for GPX4 protein expression. | Cell Death & Disease

Fig. 5: The nucleotide A575 in GPX4 (NM_002085) transcript is critical for GPX4 protein expression.

From: Depletion of the N6-Methyladenosine (m6A) reader protein IGF2BP3 induces ferroptosis in glioma by modulating the expression of GPX4

Fig. 5

A Cartoon diagram illustrating the Coding Sequence (CDS) and 3’UTR of GPX4 (NM_002085) transcript. The amino acid numbering was highlighted in magenta, and the nucleotide numbering was highlighted in red. Three potential IGF2BP3 binding sequences and the UGA codons are indicated in the diagram. B RNA pull-down assay to assess the binding potency of three RNA sequences with IGF2BP3. Three potential IGF2BP3 binding RNA sequences were synthesized with or without m6A modification and then mixed with lysate from U87 and HS683 cells. The RNA-protein complexes were pulled down, and an anti-IGF2BP3 western blot was performed to detect the interaction between IGF2BP3 and the RNA sequences. C MeRIP assay revealing m6A enrichment of different GPX4 mutants. U87 and HS683 cells were infected with lentivirus overexpressing GPX4-WT, GPX4-A575T, GPX4-A694T, or GPX4-A575/694 T. Total RNA was extracted, and mRNA was purified. The mRNA was then enriched using an anti-m6A antibody and quantified by qPCR (Paired two-tailed t-test). D-E The U87 and HS683 cells were infected with lentivirus overexpressing GPX4-WT, GPX4-A575T, GPX4-A694T, or GPX4-A575/694T. The GPX4 protein and mRNA expression levels were verified by western blot (D) and qPCR (E). F-G The U87 and HS683 cells were infected with lentivirus overexpressing GPX4-WT, GPX4-U73C, GPX4-U73C-A575T, GPX4-U73C-A694T, or GPX4-U73C-A575/694T. The GPX4 protein and mRNA expression levels were verified by western blot (F) and qPCR (G). H HEK 293 T cells were infected with lentivirus overexpressing GPX4-WT or GPX4-A575T, and treated with Actinomycin D for 0, 3, and 6 h. RNA was extracted, and qPCR was performed to verify the expression level of GPX4. The degradation lines were generated using linear regression approach. I The U87 and HS683 were infected with lentivirus overexpressing GPX4-WT or GPX4-A575T. Cytoplasmic and whole cells’ RNA was extracted, and the expression rate of GPX4 in the cytoplasm relative to the whole cells was verified using qPCR. J IGF2BP3 was overexpressed together with GPX4-WT or GPX4-A575T, the expression level of GPX4 was verified by western blot. Data are presented as the mean ± standard deviation (SD) from three independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05.

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