Fig. 5: DJ-1 depletion disrupts the generation of Hcy from SAH via impairing intracellular SAHH activity.

a–c ELISA assays for the levels of endocellular Hcy. Indicated DJ-1 KD H1299 cells a and DJ-1 KO H1299 cells b were treated with erastin (2 μM) for 12 h, and the Hcy levels were assayed. Indicated MEFs c were treated with erastin (400 nM) for 12 h, and the Hcy levels were assayed. d–f Indicated cells were deprived from Met for 24 h, followed by adding the extra SAH to the cells for 4 h, and Hcy levels we detected by ELISA. d The schematic representation of experiment design. The relative Hcy levels in indicated H1299 cells e and MEFs f are shown. g, h DJ-1 increased the enzymatic activity of SAHH. g Indicated HEK293T cells with either DJ-1 overexpression or KD (#1 sequence of DJ-1 KD was used here) were further transfected with SAHH-HA plasmids. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-HA antibody. Independent experiments are repeated three times and representative data are shown. h The activity of ectopic SAHH from cells by immunoprecipitation was assayed as mentioned in Methods section. Independent experiments were repeated three times and representative data are shown. i Western blot analysis of SAHH expression in H1299 cells with SAHH KD. Independent experiments are repeated three times and representative data are shown. j Indicated SAHH KD H1299 cells were treated with erastin (2 μM) for 12 h, and lipid ROS production was assayed by flow cytometry using C11-BODIPY. k Western blot analysis of DJ-1 and SAHH expression in indicated H1299 cells (#1 sequence of DJ-1 KD was used here). Independent experiments are repeated three times and representative data are shown. l Indicated DJ-1 KD (#1 sequence) and SAHH overexpression H1299 cells were treated with erastin (2 μM) for 12 h, and lipid ROS production was assayed. m Cell viability was assayed in indicated cells treated with 2 μM erastin for 36 h. Data shown represent mean ± SD from three independent experiments. Comparisons were made using the two-tailed, unpaired Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001; n.s., no statistic difference.