high mobility group box 1  (Chondrex Inc)

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: Mouse monoclonal antibodies against HMGB1; PCRP-HMGB1-4F10 was deposited to the DSHB by Common Fund – Protein Capture Reagents Program (DSHB Hybridoma Product PCRP-HMGB1-4F10), GAPDH; DSHB-GAPDH-2G7 was deposited to the DSHB by DSHB (DSHB Hybridoma Product DSHB-GAPDH-2G7) and LAMIN A/C MANLAC2(10F8) was deposited to the DSHB by Morris, G.E. (DSHB Hybridoma Product MANLAC2(10F8)) were purchased from Developmental Studies Hybridoma Bank (DSHB), created by the NICHD and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242.

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

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: PARylation primes HMGB1 for acetylation following rotenone treatment that is associated with decreased SIRT1 activity. A , graphical representation of post-translational modification induced by PARP1 that modulates SIRT1-HMGB1 interaction during ongoing DNA damage. B , determination of SIRT1 activity by fluorometric assay in SH-SY5Y cells following rotenone treatment in the presence or absence of PJ34 and compared with DMSO-treated controls. Results are expressed in arbitrary fluorescence units. C , immunoprecipitation analysis with anti-HMGB1 antibody to check the acetylation status of HMGB1 in the whole cell extracts of cells following rotenone treatment for 24 h and probing with pan-acetyl antibody. D and E , representative confocal images of cells stained with SIRT1 ( red ), HMGB1 ( green ) and DAPI ( blue ) following 4 ( D ) or 12 ( E ) h rotenone treatment. (scale bar = 10 μm). F , quantification of SIRT1 intensity at 12 h post rotenone treatment. (n = 100 cells for each quantification). G , representative confocal images of cells stained with SIRT1 (red), HMGB1 ( green ), and DAPI ( blue ) following 24 h rotenone treatment in the presence or absence of PJ34 or a combination of PJ34 and the SIRT1 inhibitor EX527. (scale bar = 10 μm). H , MTT assay of cells treated with rotenone for 24 h in the presence or absence of PJ34 or a combination of PJ34 and EX527. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test (∗∗ p < 0.01; ∗∗∗ p < 0.001; n = 3 biological replicates).

Article Snippet: Mouse monoclonal antibodies against HMGB1; PCRP-HMGB1-4F10 was deposited to the DSHB by Common Fund – Protein Capture Reagents Program (DSHB Hybridoma Product PCRP-HMGB1-4F10), GAPDH; DSHB-GAPDH-2G7 was deposited to the DSHB by DSHB (DSHB Hybridoma Product DSHB-GAPDH-2G7) and LAMIN A/C MANLAC2(10F8) was deposited to the DSHB by Morris, G.E. (DSHB Hybridoma Product MANLAC2(10F8)) were purchased from Developmental Studies Hybridoma Bank (DSHB), created by the NICHD and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242.

Techniques: Activity Assay, Modification, Fluorescence, Immunoprecipitation, Staining, MTT Assay, Comparison

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: HMGB1 inhibitor glycyrrhizic acid prevents rotenone-induced G2/M arrest. A , representative confocal images of cells stained with pH3S10 ( red ), HMGB1 ( green ) and DAPI ( blue ) following rotenone treatment in the presence or absence of the HMGB1 inhibitor glycyrrhizic acid (GA) (1.5 mM). (scale bar = 10 μm). B , quantification of SH-SY5Y cells with cytoplasmic enriched HMGB1 ( left panel ) and pH3S10 positive cells indicating cells arrested at G2/M ( right panel ) for each treatment condition. (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 24 h in the presence or absence of GA. GAPDH was used as cytosolic loading control and lamin A/C was used as a nuclear loading control ( upper panel ). The nuclear to cytoplasmic ratio was quantified using the lower band obtained in the cytoplasmic fraction of the gels ( lower panel ). D , representative flow cytometry histograms depicting DNA content in SH-SY5Y cells stained with propidium iodide (PI). Distinct peaks correspond to cells in the G0/G1 phase (first peak), S phase (intermediate region), and G2/M phase (second peak). SH-SY5Y cells were treated with Rot, GA or pre-incubated with GA before Rot treatment for 24 h and the data is compared with DMSO control. The quantification of cells in each phase of the cell cycle are shown in the right panel. E and F , immunoblot analysis of Cyclin B1, Securin ( E ) and BubR1 ( F ) protein levels in the whole-cell extracts of SH-SY5Y cells 24 h post-rotenone treatment in the presence or absence of GA. β-Actin was used as a loading control. G , schematic representation showing association of rotenone-induced G2/M arrest with HMGB1 nuclear exit and the effect of GA on the process. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test (∗∗∗ p < 0.01; n = 3 biological replicates).

Article Snippet: Mouse monoclonal antibodies against HMGB1; PCRP-HMGB1-4F10 was deposited to the DSHB by Common Fund – Protein Capture Reagents Program (DSHB Hybridoma Product PCRP-HMGB1-4F10), GAPDH; DSHB-GAPDH-2G7 was deposited to the DSHB by DSHB (DSHB Hybridoma Product DSHB-GAPDH-2G7) and LAMIN A/C MANLAC2(10F8) was deposited to the DSHB by Morris, G.E. (DSHB Hybridoma Product MANLAC2(10F8)) were purchased from Developmental Studies Hybridoma Bank (DSHB), created by the NICHD and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242.

Techniques: Staining, Western Blot, Control, Flow Cytometry, Incubation, Comparison

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: HMGB1 hypoacetylation mutants prevents its nuclear exit and subsequent rotenone-induced G2/M arrest. A , schematic illustration of sites mutated in HMGB1 for this study. B , representative confocal images of SH-SY5Y cells transfected with FLAG-tagged wild-type HMGB1 or its mutants as shown in ( A ) and immunostained with FLAG ( green ), pH3S10 ( red ) and DAPI ( blue ) following rotenone treatment for 24 h (scale bar = 10 μm). C , quantification of pH3S10 positive cells per transfected cell and cells with cytoplasmic enriched HMGB1 per transfected cell. (n = 50 transfected cells for each quantification). Data are presented as mean ± SD. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparison test (∗∗∗ p < 0.001).

Article Snippet: Mouse monoclonal antibodies against HMGB1; PCRP-HMGB1-4F10 was deposited to the DSHB by Common Fund – Protein Capture Reagents Program (DSHB Hybridoma Product PCRP-HMGB1-4F10), GAPDH; DSHB-GAPDH-2G7 was deposited to the DSHB by DSHB (DSHB Hybridoma Product DSHB-GAPDH-2G7) and LAMIN A/C MANLAC2(10F8) was deposited to the DSHB by Morris, G.E. (DSHB Hybridoma Product MANLAC2(10F8)) were purchased from Developmental Studies Hybridoma Bank (DSHB), created by the NICHD and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242.

Techniques: Transfection, Comparison

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-induced tubulin hyperacetylation precedes HMGB1 nuclear release. A and B , representative confocal images of SH-SY5Y cells ( A ) stained with Ac-αTubulin ( green ) and DAPI ( blue ) following rotenone treatment for 4, 6 and 12 h and PC12 cells ( B ) following rotenone treatment for 4 h (scale bar = 25 μm). C , representative confocal images in cells stained with pH3S10 ( red ), HMGB1 ( green ) and DAPI ( blue ) in SH-SY5Y cells transfected with control siRNA (siNC) or siαTAT1. (scale bar = 10 μm). D , quantification of percentage of cells with nuclear enriched HMGB1 ( upper panel ) and cells arrested at G2/M as depicted by pH3S10 positive cells ( lower panel ). (n = 100 cells for each quantification). E , representative confocal images of SH-SY5Y cells depicting the effect of GA on the acetylation of α-tubulin ( green ) induced by rotenone alongwith with pH3S10 ( red ) and DAPI ( blue ). (Scale bar = 10 μm). F , corrected total cell fluorescence (CTCF) values of α-tubulin intensity of control vs rotenone or nocodazole treated cells in the presence or absence of GA ( upper panel ). Quantification of pH3S10 positive cells ( lower panel ) similarly treated. (n = 100 cells for each quantification). G , schematic representing the effect of tubulin hyperacetylation on HMGB1 nuclear exit and subsequent G2/M arrest induced by rotenone. 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: Mouse monoclonal antibodies against HMGB1; PCRP-HMGB1-4F10 was deposited to the DSHB by Common Fund – Protein Capture Reagents Program (DSHB Hybridoma Product PCRP-HMGB1-4F10), GAPDH; DSHB-GAPDH-2G7 was deposited to the DSHB by DSHB (DSHB Hybridoma Product DSHB-GAPDH-2G7) and LAMIN A/C MANLAC2(10F8) was deposited to the DSHB by Morris, G.E. (DSHB Hybridoma Product MANLAC2(10F8)) were purchased from Developmental Studies Hybridoma Bank (DSHB), created by the NICHD and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242.

Techniques: Staining, Transfection, Control, Fluorescence, Comparison

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-induced tubulin hyperacetylation triggers mtROS-induced DNA damage. A and B , visualization of mitochondrial ROS (mtROS) production by live staining with the mitochondria-specific ROS indicator MitoSox red ( red ) and the nuclear stain Hoechst 33342 ( blue ) at 4 h ( A ) and 12 h ( B ) post-rotenone treatment in cells transfected with control siRNA (siNC) or siαTAT1. The CTCF values of MitoSox intensity are also shown. (scale bar = 10 μm). C , visualization of mtROS production by staining with MitoSox red ( red ) and the nuclear stain Hoechst 33342 ( blue ) at 12 h post-rotenone treatment in the presence of absence of the NAD(P)H oxidase inhibitor DPI (2.5 μM). (scale bar = 10 μm). D , representative confocal images of pH3S10 ( red ), HMGB1 ( green ) and DAPI ( blue ) following rotenone treatment in the presence or absence of DPI. (n = 100 cells for each quantification; scale bar=10 μm). E , quantification of cytoplasmic enriched HMGB1 ( left panel ) and pH3S10 positive cells indicating cells arrested at G2/M ( right panel ) and for each treatment condition for 100 cells. F , representative confocal images of cells stained with phospho-γH2AX (green) and DAPI ( blue ) following rotenone treatment in siNC and siα-TAT1 cells. (Scale bar = 10 μm). G , quantification of γH2AX foci/cell in rotenone treated cells transfected with siControl and α-TAT1 knockdown. (n = 50 cells for each transfection and treatment). H , schematic representation showing the effect of mtROS in inducing ds-breaks in nuclear DNA following rotenone treatment that is alleviated by blocking tubulin acetylation. 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: Mouse monoclonal antibodies against HMGB1; PCRP-HMGB1-4F10 was deposited to the DSHB by Common Fund – Protein Capture Reagents Program (DSHB Hybridoma Product PCRP-HMGB1-4F10), GAPDH; DSHB-GAPDH-2G7 was deposited to the DSHB by DSHB (DSHB Hybridoma Product DSHB-GAPDH-2G7) and LAMIN A/C MANLAC2(10F8) was deposited to the DSHB by Morris, G.E. (DSHB Hybridoma Product MANLAC2(10F8)) were purchased from Developmental Studies Hybridoma Bank (DSHB), created by the NICHD and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242.

Techniques: Staining, Transfection, Control, Knockdown, Blocking Assay, Comparison

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: Loss of nuclear HMGB1 affects the transcription of DNA damage response genes that may lead to rotenone-induced mitotic catastrophe. A , representative IGV snapshots displaying ChIP-seq binding peaks of HMGB1, reanalysed from ChIP-Atlas database, on selected DNA Damage Response (DDR) genes ATM, FEN1, XPA, GADD45A, KLF4, RAD23B. Peaks indicate strong enrichment of HMGB1 binding, suggesting potential regulatory roles at these loci. Chromosomal position and transcription start site ( red arrow ) depicted in the image. B , qRT-PCR analysis of the relative gene expression of DDR genes at 4 h ( upper panel ) or 24 h ( lower panel ) post-rotenone treatment with respect to DMSO-treated controls. β2 microglobulin (β2MG) was used as an internal control. C , representative confocal images of cells stained with phospho-γH2AX ( green ) and DAPI ( blue ) following rotenone treatment in the presence or absence of GA. (scale bar = 10 μm). D , graphical representation of the study. Data are presented as mean ± SD. Statistical significance was determined by unpaired t test with Sidak multiple comparison (∗∗ p < 0.01; ∗∗∗ p < 0.001; n = 3 biological replicates).

Article Snippet: Mouse monoclonal antibodies against HMGB1; PCRP-HMGB1-4F10 was deposited to the DSHB by Common Fund – Protein Capture Reagents Program (DSHB Hybridoma Product PCRP-HMGB1-4F10), GAPDH; DSHB-GAPDH-2G7 was deposited to the DSHB by DSHB (DSHB Hybridoma Product DSHB-GAPDH-2G7) and LAMIN A/C MANLAC2(10F8) was deposited to the DSHB by Morris, G.E. (DSHB Hybridoma Product MANLAC2(10F8)) were purchased from Developmental Studies Hybridoma Bank (DSHB), created by the NICHD and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242.

Techniques: ChIP-sequencing, Binding Assay, Quantitative RT-PCR, Gene Expression, Control, Staining, Comparison