Fig 2: SUMOylation of PIAS1 promotes its PML localization.a Distribution of SUMO sites identified on PIAS1. Three SUMO sites (K137, K237, and K315) were identified in the dataset. K137 and K238 are located in the PINIT domain, whereas K315 is located in the SP-RING domain. b HEK293-SUMOm cells were co-transfected with PIAS1-GFP-WT, PIAS1-GFP-3xKR, PIAS1-GFP-K137R, PIAS1-GFP-K238R, or PIAS1-GFP-K315R and immunofluorescence was performed with anti-PML. Scale bar: 2.5 µm. c Scatter graph showing the Pearson’s correlation coefficient for the PIAS1–PML colocalization. Data represent the mean ± SD, error bars represent SD, ns nonsignificant, *p < 0.05, **p < 0.01, Student’s t-test, n = 20 biologically independent cells/condition. Source data are provided as a Source Data file.

Fig 3: SUMOylation of vimentin is required for proper cell migration in HeLa cells.a VIM sequence indicating SUMOylation sites at Lys-439 and Lys-445 localized in the tail domain and highly conserved across six different species. b Western blotting analysis of HeLa cells transfected with an empty Flag vector as a negative control, Wild-type Flag-tagged vimentin (Flag-VIMwt), Flag-tagged vimentin K439/445R, double mutant (Flag-VIMmt), with or without Myc-PIAS1. c Flag-VIMmt expression inhibits proper cell migration and can be rescued by PIAS1 overexpression, as determined by a wound-healing assay. Scale bar: 200 µm. d Quantification of the wound-healing assay. Data represent the mean ± SD, error bars represent SD, ns nonsignificant, **p < 0.01, Student’s t-test, n = 5 biologically independent samples. Source data are provided as a Source Data file.

Fig 4: Functional effects of PIAS1 expression on HeLa cells.a HeLa cells were transfected with Myc-PIAS1 (PIAS1-OE) or Empty vector (NC-vector) for 48 h. PIAS1 overexpression efficiency was determined by western blotting. Actin was used as a loading control. b PIAS1 KO HeLa cells were generated by CRISPR/Cas9. PIAS1 KO was determined by western blotting. Actin was used as a loading control. c PIAS1 overexpression in HeLa cells significantly promotes cell growth, n = 3 biologically independent samples. d PIAS1 KO HeLa cells (PIAS1-sgRNA) showed impeded cell growth compared with the negative control cells (NC-sgRNA), n = 3 biologically independent samples. e PIAS1 overexpression increased cell migration as determined by a wound-healing assay. Scale bar: 200 µm, n = 5 biologically independent samples. f PIAS1 KO cells displayed decreased cell migration compared with the sgRNA-negative control cells, as determined by a wound-healing assay. Scale bar: 200 µm, n = 5 biologically independent samples. Data represent the mean ± SD, error bars represent SD, **p < 0.01, Student’s t-test. Source data are provided as a Source Data file.

Fig 5: PIAS1 expression is regulated by androgens at mRNA and protein levels. (a) Cells were treated with increasing concentrations of R1881 (0.1, 0.5, 1 and 10 nM) in the absence or presence of 5 µM Bicalutamide and subjected to real-time quantitative reverse transcription PCR (qRT-PCR) or western blot. Data represent mean+s.e.m. from at least three independent experiments (***P<0.001). (b, c) PIAS1 mRNA and protein expression in LNCaP, DUCaP and VCaP cells after stimulation with 1 nM R1881 for 8, 24, 48 or 72 h. Data represent mean+s.e.m. from three independent experiments (*P<0.05; **P<0.01; ***P<0.001). (d) Immunofluorescence staining for PIAS1 (red) and AR (green) after treatment of LNCaP, DUCaP and VCaP cells with 1 nM R1881 for 24 h. Scale bar=50 µm. (e) Western blot analysis of PIAS1 and AR in DUCaP and LNCaP cells following transfection with two specific AR siRNAs (siAR-1 and siAR-2; 25 nM). Quantifications of western blots represent mean+s.e.m. from at least three independent experiments (*P<0.05; ***P<0.001).