Fig 2: Cells resistant to xCT inhibition are sensitive to co-treatment with RSL3 and iFSP1.a, b Immunoblots of GPX4, FSP1, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), glutamate–cysteine ligase catalytic subunit (GCLC), glutamate–cysteine ligase modifier subunit (GCLM) in HT1080, HEK293T, A375, A549, HT29, MDA-MB-436, U-373, and UMRC2 cells (a), and HT1080, HEK293T, HLE, HLF, HepG2, and Huh7 cells (b). VCP was used as loading controls. c Cell viability of HT29, MDA-MB-436, U-373, and Huh7 cells treated with indicated concentrations of sulfasalazine, erastin, or RSL3 in the absence or presence of 5 μM iFSP1 for 24 h. Data are presented as mean ± s.d. of n = 3 wells of a 96-well plate from one representative of three independent experiments.

Fig 3: Effect of lipopolysaccharide (LPS) [10 mg/kg, intraperitoneally (i.p.), 6 h] on protein levels of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) in the spleen, lung, liver and kidney of wild-type (WT) mice. Expression levels in vehicle-treated mice for each organ are normalized as 100%. Please note that LPS induced an increase in CBS expression in the spleen and lung, an increase in CSE expression in the lung, and an increase in 3MST expression in the lung and liver. Data are shown as the means ± standard error of the mean (SEM) of n=5 determinations; *p<0.05 shows significant LPS-induced increase in the expression level of the indicated enzyme in the indicated organ.

Fig 4: Downregulation of cystathionine-β-synthase (CBS) attenuates the lipopolysaccharide (LPS)-induced increases in blood urea nitrogen (BUN) plasma levels in mice. BUN levels in wild-type (WT), CBS heterozygous (CBS+/−), cystathionine-γ-lyase (CSE) knockout (CSE−/−) and 3-mercaptopyruvate sulfurtransferase (3MST) mutant (Δ3MST) mice treated with vehicle or LPS (10 mg/kg, 6 h). LPS significantly impaired glomerular function, as evidenced by markedly increased BUN concentration; this increase was reduced in CBS+/− mice. Data are shown as mean ± standard error of the mean (SEM) of 10 animals; *p<0.05 shows significant increases in response to LPS, compared to the vehicle control; #p<0.05 shows a significant protective effect of the CBS+/− phenotype compared to WT.

Fig 5: Downregulation of cystathionine-β-synthase (CBS) or cystathionine-γ-lyase (CSE) attenuates the lipopolysaccharide (LPS)-induced increases in lung malondialdehyde (MDA), but not myeloperoxidase (MPO) levels in mice. Lung (A) MPO and (B) MDA levels in wild-type (WT), CBS heterozygous (CBS+/−), CSE knockout (CSE−/−) and 3-mercaptopyruvate sulfurtransferase (3MST) mutant (Δ3MST) mice treated with vehicle or LPS (10 mg/kg, 6 h). LPS significantly increased MPO and MDA levels; this increase was reduced in CSE−/− and CBS+/− mice. Data are shown as the means ± standard error of the mean (SEM) of 10 animals; **p<0.01 shows significant increases in response to LPS, compared to the vehicle control; #p<0.05 shows a reduction by the CSE−/− and CBS+/− phenotype compared to WT.