Fig 2: DJ-1 depletion impairs SAHH tetrameric structure via enhancing the SAHH/AHCYL1 interaction.a The scheme of BioID assay for identifying the interaction proteins of DJ-1. b The results of BioID assay. The numbers indicate the proteins identified from each group by mass spectrometry, and the top ten proteins specifically identified in biotin-treated DJ-1-BirA* samples were listed. c The interaction between AHCYL1 (the left view) or AHCYL2 (the right view), and DJ-1 in BirA* system was analyzed. HEK293T cells were transfected with indicated plasmids and treated with DMSO or 50 μM biotin for 24 h before harvested. Input and samples of IP-streptavidin were separated on SDS–PAGE gels and subjected to immunoblotting with anti-Flag and anti-DJ-1 antibodies. Independent experiments are repeated three times and representative data are shown. d The interaction between AHCYL1 and SAHH under different DJ-1 level was analyzed. Indicated HEK293T cells stably expressing AHCYL1 and SAHH were harvested for immunoprecipitation and subjected to immunoblotting with anti-Flag and anti-HA antibodies (#1 sequence of DJ-1 KD was used here). ‘s’ represents short exposure; ‘l’ represents long exposure. Independent experiments are repeated three times and representative data are shown. e Western blot analysis of DJ-1 and AHCYL1 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. f Indicated DJ-1 KD (#1 sequence) and AHCYL1 KD H1299 cells were treated with erastin (2 μM) for 12 h, and lipid ROS production was assayed. Data shown represent mean ± SD from three independent experiments. Comparisons were made using the two-tailed, unpaired Student’s t-test; *p < 0.05. g Western blot analysis of the fractions from size-exclusion chromatography experiment, showing less tetrameric SAHH complex (fraction 23–28) in DJ-1 KD H1299 cells (#1 sequence of DJ-1 KD was used here). Independent experiments are repeated three times and representative data are shown.

Fig 3: Site-specific insertion of HindIII site mediated by TALENs.(A) Schematic of the DJ-1 locus with the ssODN sequence used to introduce the HindIII sequence (underlined) into the genome in vivo. (B) One of the colonies (DS21) could be cut by the HindIII restriction enzyme. The arrow shows the resulting fragments cut by HindIII. (C) DNA sequences of the DS21 colony at the target site. The HindIII site is shown by bold uppercase letters. There is a different base before the HindIII site, as shown in lowercase letters. DS21-WT represents sequence of the mixed wild type cells in DS21 colony.

Fig 4: NRF2 is not involved in DJ-1-regulated ferroptosis.a, b Indicated DJ-1 KD H1299 cells were treated with erastin (2 μM) for 12 h. Independent experiments are repeated three times and representative data are shown. a The protein expression of NRF2, and b its known downstream genes were assayed. The relative gene expression is normalized to β-actin. c The mRNA expression of PARK7 and NRF2 in indicated H1299 cells was assayed by qRT-PCR (#1 sequence of DJ-1 KD was used here). The relative gene expression is normalized to β-actin and the error bar indicates the SD value from triplicates. d Viability of H1299 cells infected with shRNAs for 72 h (#1 sequence of DJ-1 KD was used here) and treated with erastin (1–4 μM, 24 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.

Fig 5: DJ-1 behaves as a bona fide stress granule component and interacts with specific mRNAs. a Sensitivity of stress granules to cycloheximide. HEK 293T cells were treated with 0.2 or 0.4 M sorbitol for 30 min and then incubated with cycloheximide for another 30 min in the presence of sorbitol. Cells were fixed and stained with anti-G3BP, anti-TIA1, and anti-DJ-1 antibodies. Cycloheximide causes a reduction in the number and size of G3BP positive stress granules size and a reduction in size of TIA1 positive stress granules as a function of sorbitol concentration. DJ-1 labeling shows a similar reduction after cycloheximide treatment with behaviour comparable to TIA1. ~ 600 cells were counted for each condition from two independent experiments. Data are shown as mean ± SEM. Statistical analysis by two-way ANOVA followed by post hoc analysis with a Tukey’s test; *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001. b DJ-1 interacts with specific mRNAs in HEK 293T cells. Cells were lysed and anti-DJ-1 and non-specific IgG antibodies were used to immunoprecipitate RNA bound to DJ-1 or background, respectively. The amount of transcripts associated with each sample was measured by QPCR, and levels of β-actin mRNA were used as a non-specific control. By normalizing to β-actin mRNA levels in each sample, an enrichment of the three transcripts was observed in DJ-1 IP samples compared to IgG IP samples (increase of ~ 16-fold, ~ 2.4-fold, and ~ 6.1-fold, respectively, for GPx4, eIF4B, and eIF4EBP1). Statistical significance was determined by comparing DJ-1 to IgG IP; ***P < 0.001