Fig 2: COVID-19 serum from non-survivors regulates ferroptosis via TNFR1 in HUVECs. Representative immunoblots are shown in panel (A), quantified in panel (B) (GPX4), (C) (SCL7A11), (D) (FTH1), and (E) (SAT1). All experiments were performed at least in triplicate; the box-and-whiskers graphs show the median and the 5th–95th percentiles; *: p < 0.01 vs. Survivors.

Fig 3: KDM3A allows for SRSF3 binding to SAT1 pre-mRNA. (A) MCF7 cells were transfected with nontargeting siRNA (siNT) or siSF3B1, siSRSF1/3/6 and sihnRNPF for 72 h. Total RNA was extracted and analyzed by real-time PCR for SAT1 exon X inclusion. PSI was calculated by SAT1 exon X relative to SAT1 total mRNA amount. (B) RNA-IP of SRSF3 in MCF7 cells that were transfected with nontargeting siRNA (siNT) or siKDM3A for 72 h. Real-time PCR analysis of SAT1 intron 3 (near exon X) relative to input. Values represent averages of three independent experiments ± SD. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001.

Fig 4: KDM3A regulates SAT1 alternative splicing independent of its demethylase enzymatic activity. (A) Schematic representation of SAT1 alternative splicing. Rectangles: exons, black lines: introns. (B,C) MCF7 cells were mock transfected or transfected with 1 µM SAT1 oligo for 72 h, total RNA was extracted and analyzed by real-time PCR for SAT1 exon X inclusion. PSI was calculated by SAT1 exon X relative to SAT1 total mRNA amount (B), and the cells were treated with 200 ng/mL NCS, and analyzed 8 h later using flow cytometry. The amount of cells accumulated in S-phase is shown as a fold-change relative to untreated cells (C). (D) MCF7 cells stably expressing either the empty vector, wild-type KDM3A or KDM3A mutated demethylase (mut). Total RNA was extracted and analyzed by real-time PCR for SAT1 exon X inclusion. PSI was calculated by SAT1 exon X relative to SAT1 total mRNA amount. (E) MCF7 cells were transfected with nontargeting siRNA (siNT) and siG9a or siGLP for 72 h. Total RNA was extracted and analyzed by real-time PCR for SAT1 exon X inclusion. PSI was calculated by SAT1 exon X relative to SAT1 total mRNA amount. Values represent averages of three independent experiments ± SD. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001.

Fig 5: Overexpression of nucleolin reverses the effect of indomethacin over spermidine/spermine-N1-acetyltransferase-1 (SSAT-1) levels and cell viability. H1299 cells were transfected with a Nucleolin-CFP (Cyan Fluorescent Protein) expressing vector and exposed to indomethacin. (A) Immunoblot of nucleolin in H1299 cells transfected with nucleolin-CFP vector. White arrow shows the nucleolin protein that carry the CFP. (B) Fluorescence microscopy showing the CFP fluorescence in transfected H1299 cells. (C) Representative blot of the SSAT-1 protein levels in H1299 cells transfected with the nucleolin-CFP vector and exposed to indomethacin 1 mM for 24 h. (D) Quantitation of the SSAT-1 levels in H1299 cells ells transfected with the nucleolin-CFP vector and exposed to indomethacin 1 mM for 24 h. Relative expression levels that were normalized using the control value from each individual replicate. *: p < 0.05; compared with untreated control, calculated using one-way ANOVA and Dunnet’s post-test. (E) Cell viability of H1299 cells transfected with the nucleolin-CFP vector and exposed to indomethacin 1 mM for 96 h. ***: p < 0.001 and ****: p < 0.0001, compared with the control at the same indomethacin concentration, calculated using two-way ANOVA. Data summarize the results of three independent experiments. Original Images can be found in Figure S1.