Fig 2: THAP1 Protein stability assessment. a The expression level of different mutated THAP1 proteins in transient transfected HEK cells. Wild-type and S21T THAP1 protein showed the similar expression level, the F81L THAP1 protein is lower than wild-type or S21T mutant protein, while almost no THAP1 mutant protein was detected in cells overexpression THAP1 C54Y construct. All cells were treated in the same conditions except different constructs were used for transfection. b SK-N-AS cell lines expression of the same level of different THAP1 proteins (wild-type or mutant) were selected after induced by adding doxycycline (Dox). c Diagram showed the strategy to test THAP1 protein stability. d and e THAP1 F81L protein degraded faster compared to wild-type or other mutated (S21T and L180S) THAP1 proteins at 24, 48, and 72 h. (*p < 0.01; **p < 0.001 by t test)

Fig 3: THAP1 molecular weight and subcellular distribution of THAP1. a Western blot showing a 30-kDa band and a 25-kDa band in HEK cells overexpressing wild-type THAP1 or F21L mutant THAP1 using the Proteintech antibody (12584-1-AP). A small band (around 27 kDa) was observed in HEK cells overexpressing Q154fs180X mutant THAP1. Mass spectrometry analysis detected two THAP1 peptides in the 30-kDa band (peptide 1, IHQLEQQVEK; peptide 2, LKEVVHFQK) but not in the 25-kDa band. b Wild-type and missense mutant (F81L and L180S) THAP1 protein distributed mainly in the nuclear fraction. Lamin A/C was used as marker of nuclear protein

Fig 4: THAP1 directly regulates the expression of SOD2. a Western blot analysis showed that overexpression of wild-type THAP1 but not mutant THAP1 or empty vector leads to increased expression of SNCA and SOD2. b Decreased expression of SOD2 was observed in fibroblasts from DYT6 patients, and TOR1A protein level remains normal. c ChIP-seq showed THAP1 binds to SOD2 promoter regions of different isoforms. THAP1 ChIP-seq of K562 was obtained from ENCODE database. (Pro1: promoter region of isoform 1). d Luciferase reporter analysis revealed overexpression THAP1 represses the activity of TOR1A promoter while upregulate the activity of SOD2 promoter (Pro2). (*** p < 0.001)