Fig. 4: The transsulfuration pathway (TSP) is required for QECs’ survival and ferroptosis resistance during cystine deprivation.

A Western blot analysis for TSP enzymes: CBS and CTH, under different cystine concentrations (0; 2; 20; 200 μM) for 24 h in both PECs and QECs. ACTIN was used as loading control and band intensities were normalized on control condition (PECs grown in 200 μM cystine). Statistical significance is given by one sample t-test and reported as a p-value in the figure. B Cell viability assay using crystal violet staining on PECs and QECs grown in control medium (200 μM cystine) or FMCys-Cys and treated with TSP inhibitors (TSPi, 1 mM TFA + 1 mM PAG) for 24 h. Statistical significance is given by one sample t-test and reported as a p-value in the figure. C Cell death analysis using PI staining on PECs and QECs grown in control medium (200 μM cystine) or FMCys-Cys and treated with TSP inhibitors (TSPi, 1 mM TFA + 1 mM PAG) for 24 h. Statistical significance is given by one sample t-test and reported as a p-value in the figure. D Cell viability assay using crystal violet staining on proliferating HUVECs grown over 3 days in FMCys-Cys supplemented with 1 mM Hcy, TSPi (1 mM TFA + 1 mM PAG) or both. Statistical significance is given by one sample t-test and reported as a p-value in the figure. E Lipid peroxidation levels measured by Bodipy-C11 FACS in PECs grown in FMCys-Cys and treated with 1 mM Hcy or Hcy + TSPi (1 mM TFA + 1 mM PAG) for 24 h. The ratio between oxidized and reduced Bodipy-C11 was measured and reported as fold change over control (PECs grown in 200 μM cystine, also indicated as NT). Statistical significance is given by one sample t-test and reported as adjusted p-value in the figure. F Cell viability assay using crystal violet staining on QECs grown in control medium (200 μM cystine) or FMCys-Cys for 24 h and knocked-down for CBS, CTH or both enzymes. Statistical significance is given by one sample t-test and reported as a p-value in the figure. G Western blot analysis for HO-1 under different cystine concentration (0; 2; 20; 200 μM) for 24 h in both PECs and QECs. ACTIN was used as loading control and band intensities were normalized on control condition (PECs grown in 200 μM cystine). Statistical significance is given by one sample t-test and reported as a p-value in the figure.