Journal: bioRxiv
Article Title: Biological differences in promyelocytic leukemia (PML) proteins between PML-nuclear bodies (PML-NBs) and extranuclear PML bodies (EnPBs) in arsenite-exposed cells
doi: 10.64898/2026.01.25.701640
Figure Lengend Snippet: (A) Immunofluorescence micrographs of U-2OS cells. The cells were exposed to 3 μM As 3+ for 2 or 72 h, or left untreated. After immunostaining for PML and SUMO2/3 antibodies, the cells were counterstained with DAPI (Nuc). Scale bar = 20 μm. An arrow indicates an EnPB. (B) Western blot analyses for PML and SUMO2/3 in U-2OS cells. The cells were treated with either 0.3 or 3 μM As 3+ for 2 or 72 h, or left untreated, and then lyzed with ice-cold RIPA buffer. The lysate was separated into soluble and insoluble fractions by centrifugation. (C) Western blot analyses for PML, SUMO2/3, SUMO1, and ubiquitin in U-2OS cells. The cells were pretreated with 20 μM ML792 (SUMO E1 inhibitor) or 10 μM TAK294 (ubiquitin E1 inhibitor) for 3 h, and were exposed to 3 μM As 3+ for 2 h in the presence of the inhibitor. (D) Immunofluorescence micrographs of PML -/- U-2OS cells. The cells were exposed to 3 μM As 3+ for 2 or 72 h, or left untreated. Scale bar = 20 μm. (E) Western blot analyses for PML and SUMO2/3 in the soluble fractions of wild U-2OS and PML -/- U-2OS cells. Note that SUMOylated proteins were detected in low electromobility region in both cell types. An open arrowhead indicates SUMO monomers. A right curly bracket indicates PML isoforms.
Article Snippet: U-2OS cells (HTB-96) were obtained from ATCC and cultured in McCoy’s 5A medium containing 10% heat-inactivated fetal bovine serum (FBS).
Techniques: Immunofluorescence, Immunostaining, Western Blot, Centrifugation, Ubiquitin Proteomics