Journal: The Journal of Biological Chemistry

Article Title: Tubulin hyperacetylation drives HMGB1 nuclear exit via the ROS-PARP1 axis, leading to rotenone-induced G2/M arrest

doi: 10.1016/j.jbc.2025.110695

Figure Lengend Snippet: Rotenone triggers time-dependent HMGB1 nuclear exit coupled with increased PARylation. A , representative confocal images of time-dependent HMGB1 ( green ) localization in SH-SY5Y cells treated with rotenone (5μM) or DMSO control. DAPI ( blue ) indicates nuclear staining (scale bar = 10 μm). B , quantification of SH-SY5Y cells with cytoplasmic enrichment of HMGB1 shown in ( A ). Nucleus was defined by area of DAPI. Cytosolic HMGB1 was calculated by subtracting nuclear region of interest (ROI) from the area of green channel. n = 100 cells for each quantification. C , immunoblot analysis of HMGB1 protein levels in the nuclear and cytosolic fraction of SH-SY5Y cells treated with rotenone for 2, 4, 6 and 24 h and compared with DMSO treated controls. GAPDH was used as cytosolic loading control and lamin A/C was used as a nuclear loading control ( left panel ). The nuclear to cytoplasmic ratio was quantified using the lower band obtained in the cytoplasmic fraction of the gels ( right panel ). D , co-immunoprecipitation analysis was performed to assess the PARylation status of HMGB1 in whole-cell extracts following 24 h of rotenone treatment. Cells were immunoprecipitated with an anti-HMGB1 antibody, and PAR ( upper panel ) and HMGB1 ( middle panel ; low and high exposures) levels were analyzed. Additionally, cells were immunoprecipitated with an anti-PAR antibody, and HMGB1 levels ( lower panel ) were examined. E , immunoblot analysis of HMGB1 protein levels in the nuclear and cytosolic fraction of cells treated with rotenone for 24 h in the presence or absence of the PARP inhibitor PJ34 (50μM). GAPDH was used as cytosolic loading control and lamin A/C was used as a nuclear loading control ( left panel ). The nuclear to cytoplasmic ratio was quantified using the lower band obtained in the cytoplasmic fraction of the gels ( right panel ). F , representative confocal images of HMGB1 ( green ) and DAPI (blue) in SH-SY5Y cells treated with rotenone or DMSO control in the presence of PJ34. DAPI ( blue ) indicates nuclear staining (scale bar = 10 μm). G , quantification of cells with cytoplasmic enrichment of HMGB1. n = 100 cells for each quantification. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test (∗∗∗ p < 0.001; n = 3 biological replicates).

Article Snippet: For determining the level of PARylation, anti-PAR antibody (#MCA-1480) and mouse monoclonal antibody for β-actin (#MCA5775GA) was obtained from Bio-Rad.

Techniques: Control, Staining, Western Blot, Immunoprecipitation, Comparison