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

Journal: Cells

Article Title: Characterization of PARP6 Function in Knockout Mice and Patients with Developmental Delay

doi: 10.3390/cells10061289

Figure Lengend Snippet: Identification of CRISPR/Cas9-mediated genome modification of Parp6 in mice by sequencing and genotyping analysis. ( A ) sgRNA target site (red) followed by the PAM sequence (blue) in the Parp6 gene. ( B ) Schematic representation of the domain structure of Parp6 WT : additional sequence found in neuronal cells (blue box), catalytic domain (gray box), sgRNA target site and CRISPR-derived changes (Δ 5bp del + 1bp insert, red box), and frameshift mutation leading to changed residues and an early stop codon (orange box) resulting in a truncated Parp6 (Parp6 TR ). ( C ) Mouse genotyping shows upper 1495bp band representing the indel allele (Parp6 TR ), and lower 743bd band representing the wild-type Parp6 WT allele. Parp6 HT (heterozygous) shows both alleles.

Article Snippet: Briefly, clarified lysates of HEK-293 transfected with the different Parp6 expression constructs were immunoprecipitated with GFP-trap dynabeads (Chromotek, Planegg-Martinsried, Germany) for 1 h at 4 °C with rotation.

Techniques: CRISPR, Modification, Sequencing, Derivative Assay, Mutagenesis

Journal: Cells

Article Title: Characterization of PARP6 Function in Knockout Mice and Patients with Developmental Delay

doi: 10.3390/cells10061289

Figure Lengend Snippet: CRISPR-generated Parp6 mutation significantly reduces the levels of full-length Parp6 transcripts and protein compared to Parp6 wild-type and heterozygous animals. ( A ) RT-PCR image of Parp6 WT and Parp6 TR transcripts levels relative to mouse HPRT control, showing a significant reduction in 5′Parp6 transcripts in the CRISPR mutants compared to Parp6 WT and Parp6 HT (one-way ANOVA, p < 0.0001, intensity of bands, n = 4). ( B ) Validation of Parp6 antibody using lysates from cortical neuronal cultures transduced with shParp6 or shLacZ control. Parp6 protein levels relative to β-actin show a significant 97.3% reduction in PARP6 in the shParp6 cortical neurons compared to shLacZ transduced neurons (shLacZ = 0.646 ± 0.067, shPARP6 = 0.017 ± 0.008; t -test p < 0.0007, n = 3). ( C ) Western blot showing expression of full-length Parp6 relative to β-actin (Parp6 WT = 2.037 ± 0.037, Parp6 HT = 2.141 ± 0.371, and Parp6 TR = 0.320 ± 0.157, mean ± SEM, n = 2) indicating a significant reduction in Parp6 protein in the CRISPR mutants. (*) indicates where the truncated Parp6 protein would be (predicted 57 KDa).

Article Snippet: Briefly, clarified lysates of HEK-293 transfected with the different Parp6 expression constructs were immunoprecipitated with GFP-trap dynabeads (Chromotek, Planegg-Martinsried, Germany) for 1 h at 4 °C with rotation.

Techniques: CRISPR, Generated, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Control, Biomarker Discovery, Transduction, Western Blot, Expressing

Journal: Cells

Article Title: Characterization of PARP6 Function in Knockout Mice and Patients with Developmental Delay

doi: 10.3390/cells10061289

Figure Lengend Snippet: While Parp6 TR E18 embryos are indistinguishable from Parp6 WT and Parp6 HT embryos, P21 Parp6 TR mice are significantly smaller compared to Parp6 WT and Parp6 HT animals. ( A ) Representative image of Parp6 WT , Parp6 HT , and Parp6 TR embryos age E18, showing that they are indistinguishable from each other at this age. Scale bar: 1 cm. ( B ) Body weights of E18 embryos from two separate litters showing no significant differences (one-way ANOVA Litter 1 p = 0.4087 (n = 8), Litter 2 p = 0.1027 (n = 9). Combined, Parp6 TR and Parp6 HT are 97% and 104% of Parp6 WT , respectively. ( C ) Body weights of P21 animals (one-way ANOVA p = 0.0066; Parp6 TR and Parp6 HT are 31% ( p < 0.001) and 80% ( p < 0.05) from Parp6 WT , respectively).

Article Snippet: Briefly, clarified lysates of HEK-293 transfected with the different Parp6 expression constructs were immunoprecipitated with GFP-trap dynabeads (Chromotek, Planegg-Martinsried, Germany) for 1 h at 4 °C with rotation.

Techniques:

Journal: Cells

Article Title: Characterization of PARP6 Function in Knockout Mice and Patients with Developmental Delay

doi: 10.3390/cells10061289

Figure Lengend Snippet: Parp6 TR newborns are indistinguishable from Parp6 WT animals. ( A ) Representative image of newborns that are under 30 min old, note the stomachs of all three animals with milk. Scale bar: 0.5 cm. ( B ) Representative image of newborns just under 60 min old, note the one animal (Parp6 TR ) that turned gray (*). Scale bar= 0.5cm.

Article Snippet: Briefly, clarified lysates of HEK-293 transfected with the different Parp6 expression constructs were immunoprecipitated with GFP-trap dynabeads (Chromotek, Planegg-Martinsried, Germany) for 1 h at 4 °C with rotation.

Techniques:

Journal: Cells

Article Title: Characterization of PARP6 Function in Knockout Mice and Patients with Developmental Delay

doi: 10.3390/cells10061289

Figure Lengend Snippet: Innervation of rib cage and diaphragm of Parp6 TR is indistinguishable from Parp6 WT newborns, while Parp6 TR animals show less air in their lungs after 60 min of being born. ( A ) Representative images of rib cages (top) and diaphragms (bottom) of E18 Parp6 WT and Parp6 TR labeled with β-tubulin showing their innervation patterns (top). ( B ) Lung sections of Parp6 WT and Parp6 TR newborns (under 60 min old) stained with H&E showing size and proportion of open alveolar spaces (air, top). Scale bar = 25 µm. ( C ) Representative distribution of individual alveolar air spaces (µm 2 ), showing that individual air spaces are significantly fewer and smaller in Parp6 TR compared to Parp6 WT ( t -test p = 0.0029, **). ( D ) Percentage of total alveolar space of Parp6 WT vs. Parp6 TR ( t -test p < 0.0001, ***). n = 389 and 288 for Parp6 WT and Parp6 TR , respectively (bottom). n = 4 images for each genotype.

Article Snippet: Briefly, clarified lysates of HEK-293 transfected with the different Parp6 expression constructs were immunoprecipitated with GFP-trap dynabeads (Chromotek, Planegg-Martinsried, Germany) for 1 h at 4 °C with rotation.

Techniques: Labeling, Staining

Journal: Cells

Article Title: Characterization of PARP6 Function in Knockout Mice and Patients with Developmental Delay

doi: 10.3390/cells10061289

Figure Lengend Snippet: Genetic background of PARP6 mutations. Age is shown in years as (age at diagnosis/current age), zygosity is based on allelic variant frequency, ND = not determined.

Article Snippet: Briefly, clarified lysates of HEK-293 transfected with the different Parp6 expression constructs were immunoprecipitated with GFP-trap dynabeads (Chromotek, Planegg-Martinsried, Germany) for 1 h at 4 °C with rotation.

Techniques: Biomarker Discovery, Variant Assay

Journal: Cells

Article Title: Characterization of PARP6 Function in Knockout Mice and Patients with Developmental Delay

doi: 10.3390/cells10061289

Figure Lengend Snippet: Parp6 TR and Parp6 C563R expressing hippocampal neurons showed decreased MARylation activity of Parp6, as well as reduced dendritic branching. ( A ) Schematic representation of Parp6 WT showing the position of the extra neuronal sequence (blue), the catalytic domain (gray), and the location of the identified clinical point mutations PARP6 P111L , PARP6 H256Y , PARP6 R485H/C , and PARP6 C563R found in patients. ( B ) Western blot of the auto mono-MARylation of Parp6 WT , Parp6 C563R , Parp6 R483H , and Parp6 TR . (*) indicates GFP-Parp6 protein (left), and quantification of ADPr activity of the different Parp6 constructs (right). ( C ) Parp6 R485H increased, whereas Parp6 C563R and Parp6 TR significantly decreased dendritic complexity in primary hippocampal neurons. E18 primary rat hippocampal neurons were co-transfected with mApple (filler) and GFP-Parp6WT (control), GFP-Parp6 C563R , GFP-Parp6 R485H , or GFP-Parp6 TR on DIV7 and fixed on DIV12. Shown are representative binary images generated from fluorescent images. Scale bar, 20 μm. ( D ) Quantification of results in ( C ) using Sholl analysis. Error bars represent SEM. * p < 0.05, ** p < 0.001 (one-way ANOVA followed by Tukey’s HSD test) compared to Parp6 WT .

Article Snippet: Briefly, clarified lysates of HEK-293 transfected with the different Parp6 expression constructs were immunoprecipitated with GFP-trap dynabeads (Chromotek, Planegg-Martinsried, Germany) for 1 h at 4 °C with rotation.

Techniques: Expressing, Activity Assay, Sequencing, Western Blot, Construct, Transfection, Control, Generated