Journal: Science Advances

Article Title: TOP2B modulates DNA supercoiling and chromatin contacts during transcriptional induction

doi: 10.1126/sciadv.adu6524

Figure Lengend Snippet: ( A ) Immunoblot of small ubiquitin-like modifier 2/3 (SUMO2/3), TOP2B, TOP2A, and ERα in MCF7 extracts. Input and IPs using anti-ERα, -TOP2B, -TOP2A, and immunoglobulin G (IgG). ( B and C ) Immunoblot of TOP2B, TOP2A, ZATT, and ERα in MCF7 extracts. Input and IP as indicated. ( D ) SUMO2/3 and α-tubulin (TUB) immunoblot in inputs and anti-ERα IPs from MCF7 cells transfected with control (siC) or ZATT-specific siRNAs (siZATT). Relative SUMO2/3 signal normalized to input tubulin is shown below. ( E ) ICEIP qPCR quantifying TOP2B activity at GREB1 enhancer and promoter in hormone-depleted MCF7 cells transfected with siC or siZATT, under control (Veh) or E2-treated conditions (10 nM for 45 min). Cells pretreated with VP16 (100 μM for 30 min). Mean + SEM of ≥ three experiments; two-way ANOVA. ( F ) Gel electrophoresis of negatively supercoiled pBR322 plasmid incubated with recombinant TOP2B (28 nM) with or without ZATT (500 nM). Positions of relaxed (R), supercoiled (−), and topoisomer bands are indicated. ( G ) Annotation of ZATT ChIP-seq peaks. ( H ) Metaplot of ZATT ChIP-seq enrichment (reference-corrected RPGC) along E2-responsive genes in hormone-depleted MCF7 cells treated with Veh or E2 (10 nM for 45 min). Right: Average FC (median of log 2 ) of ZATT ChIP-seq enrichment upon E2 treatment at unresponsive and E2-responsive genes. Statistics by t test. ( I ) As in (H) at enhancers. ( J ) Quantification of ZATT ChIP-seq at E2-induced promoters with or without ERα recruitment (logFC > 1.5). ( K ) Genome browser view of ZATT and ERα ChIP-seq signals at GREB1 locus. Gray and green boxes indicate enhancer [as determined by previous ERα ChIA-PET experiments ] and gene, respectively. ( L ) RNA Pol II ChIP-seq FC upon E2 treatment (10 nM for 45 min) at E2-induced genes and enhancers in hormone-depleted MCF7 cells with siC or siZATT. Mean of two replicates; t test. Box plots: 10 to 90 percentile. Only significant differences shown.

Article Snippet: The reaction mixture consisted of 75 nM TOP2B (Inspiralis, HTB201), 3 μM SUMO2 (SignalChem, S294-31H-200), 1 μM UBC9, 100 nM SUMO E1 mix (SAE1/UBA2, Enzo, BML-UW9330-0025), and 10 nM ZATT.

Techniques: Western Blot, Ubiquitin Proteomics, Transfection, Control, Activity Assay, Nucleic Acid Electrophoresis, Plasmid Preparation, Incubation, Recombinant, ChIP-sequencing, ChIA Pet Assay

Journal: Frontiers in Psychiatry

Article Title: SENP3/FIS1-regulated PFC neural mitochondrial fragmentation underlies the mechanism of electroacupuncture attenuating depressive behavior in CUMS mice

doi: 10.3389/fpsyt.2025.1645757

Figure Lengend Snippet: EA reduces mitochondrial fragmentation via the SENP3/FIS1 pathway. (A) Western blotting images of PFC SUMO2/3, SENP3; (B) Histogram of the relative expression of SUMO2/3; (C) Histogram of the relative expression of SENP3; (D) Representative graphs of PFC FIS1 and SENP3 co-localization in IF. *P<0.05, **P<0.01.

Article Snippet: After blocking with BSA/TBST at room temperature for 3 h, the membranes were incubated with primary antibodies, including DRP1 (1:1500, 12957-1-Ap, Proteintech, Chicago, USA), MFF (1:4000, 66527-1-Ig, Proteintech), FIS1 (1:1000, SAB2702049, Sigma-Aldrich), SUMO2/3 (1:1500, 11251-1-Ap, Proteintech), SENP3 (1:1000, 17659-1-Ap, Proteintech), and GAPDH (1:3000, HRP-60004, Proteintech), overnight at 4°C.

Techniques: Western Blot, Expressing, IF-P

Journal: mBio

Article Title: EBNA1 SUMOylation by PIAS1 suppresses EBV lytic replication and enhances episome maintenance

doi: 10.1128/mbio.02639-25

Figure Lengend Snippet: PIAS1 enhances EBNA1 SUMOylation both in vivo and in vitro . ( A ) HEK-293T cells were transfected with plasmids encoding Halo-V5-EBNA1, Halo-PIAS1, and His-SUMO2. Whole-cell lysates (input) were analyzed by WB using antibodies against SUMO2/3, PIAS1, V5, and β-actin. SUMOylated proteins are indicated by brackets. EBNA1 was immunoprecipitated using anti-V5 magnetic beads, followed by WB analysis with antibodies as indicated. Arrows denote SUMOylated EBNA1. ( B ) In vitro SUMOylation assay was conducted using a combination of purified proteins, including E1, E2, SUMO2, PIAS1, and the substrate V5-EBNA1, as specified. The reaction was stopped by adding 2× SDS-PAGE loading buffer, followed by WB analysis using anti-V5-HRP antibody. SUMOylated EBNA1 is indicated by brackets. ( C ) WB analysis of PIAS1 and β-actin expression in non-targeting control (NC) and PIAS1-depleted (sg-PIAS1) Akata (EBV+) cells. ( D ) Control (NC) and PIAS1-depleted (sg-PIAS1) Akata (EBV+) cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature, followed by incubation with mouse anti-EBNA1 and rabbit anti-SUMO2/3 antibodies. PLA probes were subsequently added for ligation and amplification. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and visualized using a Nikon AXR confocal microscope. The close proximity between EBNA1 and SUMO2/3 is indicated by red fluorescent PLA signals.

Article Snippet: Briefly, cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at room temperature for 1 h, then incubated with PBS control or a mixture of mouse anti-EBNA1 (Cat. #sc-81581, Santa Cruz) and rabbit anti-PIAS1 (Cat. #ab77231, Abcam) or rabbit anti-SUMO2/3 (Cat. # 11251-1-AP, Proteintech) antibodies (1:50 dilution in 3% BSA) at 4°C overnight.

Techniques: In Vivo, In Vitro, Transfection, Immunoprecipitation, Magnetic Beads, Purification, SDS Page, Expressing, Control, Saline, Incubation, Ligation, Amplification, Staining, Microscopy