Journal: bioRxiv

Article Title: ROS Impair Mitophagy via PARylation of PINK1

doi: 10.64898/2026.06.18.733102

Figure Lengend Snippet: (A) PARP1 activation induced by DNA damage depletes intracellular ATP and NAD + . (B) Wild-type (WT) and PARP1 knockout (KO) YFP-Parkin-HeLa cells were treated with or without O/A (1 µM/1 µM), 6-OHDA (100 µM) or in combination for 10 min and harvested for immunoblotting. (C) Intracellular ATP levels response to 6-OHDA treatment over time. YFP-Parkin-HeLa cells treated with or without O/A (1 µM/1 µM), 6-OHDA (100 µM) or in combination for different times. ATP levels were measured via luciferase assay. bars, ± SD of 3 independent experiments; ****, P < 0.0001 (Two-way ANOVA test). (D) Intracellular NAD + levels response to 6-OHDA treatment over time. YFP-Parkin-HeLa cells were treated as in (C). NAD + levels were quantified via WST-8. Statistical analysis was performed as in (C). (E-F) Intracellular ATP and NAD + levels response to Olaparib. Cells were pre-treated by Olaparib for 1.5 h and then treated with O/A (1 µM/1 µM) and 6-OHDA (100 µM) for 2 h. bars, ± SD of 3 independent experiments; ns, no significance, ***, P < 0.001; ****, P < 0.0001 (Student’s t-test).

Article Snippet: The indicated amount of purified PARP1 protein (MCE, HY-P74652) and PINK1 protein (origene, TP762427) were incubated in reaction buffer (50 mM Tris-HCl pH 7.5, 4 mM MgCl 2 , 20 mM NaCl and 250 μM fresh DTT) with 1 mM NAD + and 400 ng sheared salmon sperm DNA at 25°C for 2 h. PARylation reactions were stopped by adding 1×Laemmli sample buffer and boiling at 95°C for 10 min.

Techniques: Activation Assay, Knock-Out, Western Blot, Luciferase

Journal: bioRxiv

Article Title: ROS Impair Mitophagy via PARylation of PINK1

doi: 10.64898/2026.06.18.733102

Figure Lengend Snippet: (A) YFP-Parkin-HeLa cells were treated with or without O/A (1 µM/1 µM), 6-OHDA (100 µM) or in combination, and harvested at the indicated time points for immunoblotting. (B) YFP-Parkin-HeLa cells were treated with O/A (1 µM/1 µM) and different concentrations of 6-OHDA. Cells were harvested for immunoblotting analysis with the indicated antibodies. (C) YFP-Parkin-HeLa cells were pre-treated with Olaparib (1 µM) for 1.5 h and then treated with O/A (1 µM/1 µM) and 6-OHDA (100 µM) for 6 h. Cells were harvested for immunoblotting analysis with the indicated antibodies. (D) mt-Keima-HeLa cells were treated with O/A (1 µM/1 µM) and 6-OHDA (100 µM) in the presence or absence of Olaparib (1 µM) for 4 h, followed by flow cytometry analysis at 405 nm (neutral pH) and 561 nm (acidic pH) excitation. (E) Mitophagy level was quantified in mt-Keima-HeLa cells following treatment as described in (D). bars, ± SD of 3 independent experiments; ****, P < 0.0001 (Two-way ANOVA test). (F) YFP-Parkin-HeLa cells were treated as in (D) for 1 h and imaged under confocal microscope. Scale bar: 20 μm. (G) WT and PARP1 KD YFP-Parkin-HeLa cells were treated with or without O/A (1 µM/1 µM), 6-OHDA (100 µM) or in combination for 6 h and harvested for immunoblotting. (H) WT and PARP1 KO YFP-Parkin-HeLa cells were treated as in (G) and harvested for immunoblotting.

Article Snippet: The indicated amount of purified PARP1 protein (MCE, HY-P74652) and PINK1 protein (origene, TP762427) were incubated in reaction buffer (50 mM Tris-HCl pH 7.5, 4 mM MgCl 2 , 20 mM NaCl and 250 μM fresh DTT) with 1 mM NAD + and 400 ng sheared salmon sperm DNA at 25°C for 2 h. PARylation reactions were stopped by adding 1×Laemmli sample buffer and boiling at 95°C for 10 min.

Techniques: Western Blot, Flow Cytometry, Microscopy

Journal: bioRxiv

Article Title: ROS Impair Mitophagy via PARylation of PINK1

doi: 10.64898/2026.06.18.733102

Figure Lengend Snippet: ROS-induced DNA damage activates PARP1, contributes to PARylation and ATP/NAD + reduction. PARP1 mediates PARylation of PINK1, impairing its function and further inhibiting mitophagy.

Article Snippet: The indicated amount of purified PARP1 protein (MCE, HY-P74652) and PINK1 protein (origene, TP762427) were incubated in reaction buffer (50 mM Tris-HCl pH 7.5, 4 mM MgCl 2 , 20 mM NaCl and 250 μM fresh DTT) with 1 mM NAD + and 400 ng sheared salmon sperm DNA at 25°C for 2 h. PARylation reactions were stopped by adding 1×Laemmli sample buffer and boiling at 95°C for 10 min.

Techniques:

Journal: Journal of cell science

Article Title: Cancer metabolism in radiation sensitization: complementary roles of O-GlcNAc transferase (OGT) and PARP1

doi: 10.1242/jcs.264322

Figure Lengend Snippet: A . The nutritional sensing Hexosamine Biosynthetic Pathway (HBP) integrates availability and metabolism of glucose (fructose-6-phosphate, F6P), amino acids (glutamine), fatty acids (Acetyl-CoA) and nucleotides (UTP) in generating UDP-GlcNAc, a nucleotide sugar donor for protein O-GlcNAcylation. Feeding cells with GlcNAc increases UDP-GlcNAc biosynthesis without substantially changing cell metabolism. The enzyme O-GlcNAc transferase (OGT) catalyzes the addition of O-GlcNAc to serine or threonine residues of proteins, while O-GlcNAcase (OGA) removes this modification. OSMI-1 and ST060266 are small-molecule inhibitors of OGT, while PUGNAc inhibits OGA. B-C . Western blots confirming knockdown of OGT and OGA in cells stably expressing doxycycline-inducible shRNAmir (shOGT or shOGA) compared to scrambled control (shScr) after 48 hours of doxycycline (1 μg/ml) treatment. D. Western blot showing the effects of OGT and OGA knockdown on global O-GlcNAcylation. shScr, shOGT, and shOGA cells were treated with 1 μg/ml doxycycline for 48 hours. As a control, shScr cells were also treated overnight with 50 μM PUGNAc (an OGA inhibitor) to increase O-GlcNAcylation. For B-D, total cell lysates were analyzed, and β-actin was probed as a loading control. E . Compared to untargeted control (shScr), silencing OGT (shOGT) or OGA (shOGA) modulates the DSB repair defect in MCF7 cells after irradiation with or without PARP inhibitor. shRNAs were induced for 48 h with 1 μg/ml doxycycline, and cells treated for 1 h with 0 or 10 μM veliparib before 6 Gy irradiation. Neutral comet assays were performed 24 h after 6 Gy. Plots show the percentage of tail DNA for individual nuclei (circles). Similarly, silencing OGT or OGA modulates γH2AX foci persistence in MCF7 cells after irradiation with or without PARP inhibitor. F . Representative images demonstrating effects of shScr, shOGT, and shOGA at 24 h after 6 Gy ± 10 μM veliparib on persistence of γH2AX foci (pseudo-colored in red) with DNA counterstained using DAPI (blue), with overlays. G . Plots show the number of γH2AX foci per nucleus in individual cells as shown in F . Silencing OGT or OGA also modulates 53BP1 foci persistence in MCF7 cells after irradiation with or without PARP inhibitor. H . Effects of shScr, shOGT, and shOGA at 24 h after 6 Gy ± 10 μM veliparib on 53BP1 foci (pseudo-colored in green) with DAPI (blue). I . Plots show the number of 53BP1 foci per nucleus in individual cells as shown in H . For images, scale bar = 20 μm; inset is mean ± SEM foci per nucleus. For plots, red bars indicate mean ± SEM; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05 (unpaired t-test).

Article Snippet: To generate MCF7 cell lines deficient in PARP1 or EZH2, sets of three pre-designed single-guide RNAs (sgRNAs) targeting human PARP1 or EZH2, along with recombinant SpCas9 nuclease, were purchased from Synthego.

Techniques: Modification, Western Blot, Knockdown, Stable Transfection, Expressing, Control, Irradiation

Journal: Journal of cell science

Article Title: Cancer metabolism in radiation sensitization: complementary roles of O-GlcNAc transferase (OGT) and PARP1

doi: 10.1242/jcs.264322

Figure Lengend Snippet: A , B . Western blot validation of PARP1 ( A ) and EZH2 ( B ) knockout (KO) pools. MCF7 cells were electroporated with CRISPR RNP formed with sets of three sgRNAs targeting the PARP1 or EZH2 initiation codons or three non-targeted guides used as WT control. C-E . MCF7 WT ( C ), PARP1-KO ( D ), and EZH2-KO ( E ) cells were incubated with 10 μM of BrdU for 24 h, followed by 1 h treatment before 6 Gy with 0 or 10 μM veliparib ± 50 μM PUGNAc. After 24 h, cells were fixed and stained with anti-BrdU under non-denaturing conditions (upper) or with anti-RPA (lower). F , G . Plots of BrdU ( F ) or RPA ( G ) foci per nucleus for cells treated as in C - E . For images, scale bar = 20 μm; inset is mean ± SEM foci per nucleus. For plots, red bars indicate mean ± SEM; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05 (unpaired t-test).

Article Snippet: To generate MCF7 cell lines deficient in PARP1 or EZH2, sets of three pre-designed single-guide RNAs (sgRNAs) targeting human PARP1 or EZH2, along with recombinant SpCas9 nuclease, were purchased from Synthego.

Techniques: Western Blot, Biomarker Discovery, Knock-Out, CRISPR, Control, Incubation, Staining

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Affinity-purified immunoprecipitates from HEK293T cells via anti-Flag immunoprecipitation (IP) were analyzed by mass spectrometry to identify unique peptides associated with Flag-tagged FOXN3. The sequences of the PARP1 peptides linked to FOXN3 are presented. ( B ) A co-IP assay was carried out in A549 cells to examine the endogenous association between FOXN3 and PARP1. ( C ) An anti-Flag co-IP assay was performed in A549 cells stably expressing Flag-tagged FOXN3 to assess the association between FOXN3 and PARP1 upon TGF-β treatment (10 ng/ml) at the indicated time points [0, 16, 24, and 48 hours (hr)]. IB, immunoblot. The cells were treated with MG-132 (20 μM, 4 hours) before collection. ( D ) An immunofluorescence assay was performed in A549 cells to assess the colocalization of FOXN3 and PARP1. Scale bar, 10 μm. ( E ) A deletion-mapping assay was performed in HEK293T cells to define the region within FOXN3 associated with PARP1. Flag-tagged FOXN3 WT or its various mutants were cotransfected with HA-tagged PARP1 into HEK293T cells (ΔNTD: Δ1-113aa; ΔForkhead: Δ114-199aa; ΔCTD: Δ200-490aa). ( F ) An in vitro GST pull-down assay was performed to examine the direct interaction between bacterially expressed GST-tagged FOXN3 and His-tagged PARP1 purified from Sf9 insect cells. ( G ) Venn diagrams illustrating the overlapping genes cotargeted by FOXN3 and PARP1 in A549 cells, as determined via CUT&Tag data analysis. ( H ) The normalized distribution of reads for the FOXN3 and PARP1 peaks at the transcription start site (TSS) is presented. ( I ) The binding profiles of FOXN3 and PARP1 at representative Smad response genes in A549 cells were analyzed via CUT&Tag data. The blotting data for (B), (C), (E), and (F) were quantified as the means from three independent experiments using ImageJ software. The data for (C) and (E) were analyzed using one-way ANOVA. ** P < 0.01 and *** P < 0.001.

Article Snippet: The PARP1 protein purified from Sf9 insect cells was obtained from MedChemExpress (no. HY- P74652 ).

Techniques: Affinity Purification, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Mapping Assay, In Vitro, Pull Down Assay, Purification, Binding Assay, Software