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: Oncology Reports

Article Title: Sphingosine-1-phosphate receptor 1 enhances olfactory receptor 51E1-mediated inhibition of proliferation via Src/JNK signaling in prostate cancer cells

doi: 10.3892/or.2026.9103

Figure Lengend Snippet: S1PR1 enhances OR51E1-mediated cytotoxicity and apoptosis in LNCaP cells. (A) LNCaP cells were transfected with an empty vector, S1PR1, or DRD2. After 48 h, cells were treated with NA (0.5 mM) for an additional 48 h, and cell viability was measured using the Cell Counting Kit-8 assay. (B) LNCaP cells were transduced with lentivirus encoding S1PR1 and selected with puromycin (1 µg/ml). Cell viability of parental LNCaP and LNCaP-S1PR1 cells was measured 48 h after treatment with increasing concentrations of NA (0.1–1 mM). (C) Apoptosis was assessed in LNCaP cells treated with NA for 48 h using annexin V/PI staining. Apoptotic cells were quantified as the percentage of annexin V-positive cells (early apoptosis) and annexin V/PI double-positive cells (late apoptosis) relative to the total cell population. (D) Caspase-3 activation was measured by flow cytometry following NA treatment. (E) PARP1 cleavage was analyzed by western blotting in NA-treated cells. Band intensities were quantified using ImageJ software. Data represent the mean ± SEM of at least three independent experiments. Statistical significance was determined using an unpaired Student's t-test. *P<0.05, **P<0.01 and ***P<0.001. S1PR1, sphingosine-1-phosphate receptor 1; OR51E1, olfactory receptor 51E1; DRD2, dopamine receptor D2; NA, nonanoic acid; PI, propidium iodide; ns, not significant.

Article Snippet: Anti-S1PR1 (cat. no. 55133-1-AP) and anti-PARP1 (cat. no. 51-6639GR) antibodies were purchased from Proteintech Group, Inc. and BD Biosciences, respectively.

Techniques: Transfection, Plasmid Preparation, Cell Counting, Transduction, Staining, Activation Assay, Flow Cytometry, Western Blot, Software, Olfactory

Journal: iScience

Article Title: PBX3 regulates mast cell parthanatos via TOP2A mediated DNA damage in allergic rhinitis

doi: 10.1016/j.isci.2026.115426

Figure Lengend Snippet: Regulatory role of TOP2A in mast cell apoptosis, parthanatos pathway, and inflammatory response In vitro experimental groups: CON, model, Model+OE-NC, Model+OE-TOP2A, Model+si-NC, Model+si-TOP2A. (A and B) RT-qPCR and western blot analyses were conducted to assess the mRNA and protein expression levels of TOP2A and PARP-1 in mast cells. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the CON or Model+OE-NC or Model+si-NC group. (C) TUNEL staining was performed to evaluate and visualize cell apoptosis. ∗ p < 0.05, ∗∗ p < 0.01 vs. the CON or Model+OE-NC or Model+si-NC group; scale bars, 50 μm. (D) Cell proliferation activity in each group was measured using the CCK-8 assay. ∗∗ p < 0.01 vs. the CON or Model+OE-NC or Model+si-NC group. (E and F) MMP was assessed using the JC-1 assay to evaluate mitochondrial function. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the CON or Model+OE-NC or Model+si-NC group. (G) Western blotting was used to examine the protein levels of mitochondrial AIF (Mito-AIF), cytoplasmic AIF (Cyto-AIF), and nuclear AIF (Nucleo-AIF), along with visual analysis. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the CON or Model+OE-NC or Model+si-NC group. (H and I) ELISA was employed to determine the concentrations of β-hexosaminidase, Histamine, IL-4, IL-5, and IFN-γ. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the CON or Model+OE-NC or Model+si-NC group. Data are represented as mean ± SEM ( n = 3 per group) from independent biological replicates. One-way ANOVA with Tukey’s post hoc correction for (A–D) and (F–I).

Article Snippet: Cells were then incubated overnight at 4°C with a primary antibody against PARP-1 (PB9309, BOSTER, Pleasanton, CA, USA, 1:100).

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Expressing, TUNEL Assay, Staining, Activity Assay, CCK-8 Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: PBX3 regulates mast cell parthanatos via TOP2A mediated DNA damage in allergic rhinitis

doi: 10.1016/j.isci.2026.115426

Figure Lengend Snippet: Effects of TOP2A on DNA damage, parthanatos regulation, and degranulation in mast cells Experimental groups: Model+OE-NC, Model+OE-TOP2A, Model+si-NC, Model+si-TOP2A. (A) Western blotting was employed to determine the protein expression levels of γ-H2AX. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+si-NC group. (B and C) IF staining was performed to assess the expression of the DNA damage marker γ-H2AX in mast cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+si-NC group; scale bar = 50 μm. (D) Cellular senescence was evaluated using an SA-β-gal staining kit. ∗ p < 0.05, ∗∗ p < 0.01 vs. the Model+OE-NC or Model+si-NC group; scale bars, 50 μm. Experimental groups: Model+OE-NC, Model+OE-TOP2A, Model+OE-TOP2A + DOX. (E and F) RT-qPCR and western blot analyses were conducted to detect the gene and protein expression levels of TOP2A and PARP-1 under different treatment conditions. ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+OE-TOP2A group; ns, not significant ( p ≥ 0.05). (G) Western blotting was employed to determine the protein expression levels of γ-H2AX. ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+OE-TOP2A group. (H) IF staining was used to evaluate γ-H2AX expression as a marker of DNA damage. ∗ p < 0.05, ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+OE-TOP2A group; scale bars, 50 μm. (I) SA-β-gal staining was used to assess cellular senescence. ∗ p < 0.05, ∗∗ p < 0.01 vs. the Model+OE-NC or Model+OE-TOP2A group; scale bars, 50 μm. (J) Western blot analysis was used to determine the protein expression levels of Mito-AIF, Cyto-AIF, and Nucleo-AIF. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+OE-TOP2A group. (K and L) ELISA assays were carried out to measure the levels of β-hexosaminidase, Histamine, IL-4, IL-5, and IFN-γ. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+OE-TOP2A group. Data are represented as mean ± SEM ( n = 3 per group) from independent biological replicates. one-way ANOVA with Tukey’s post hoc correction for (A and C–L).

Article Snippet: Cells were then incubated overnight at 4°C with a primary antibody against PARP-1 (PB9309, BOSTER, Pleasanton, CA, USA, 1:100).

Techniques: Western Blot, Expressing, Staining, Marker, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: PBX3 regulates mast cell parthanatos via TOP2A mediated DNA damage in allergic rhinitis

doi: 10.1016/j.isci.2026.115426

Figure Lengend Snippet: Effects of PBX3 silencing on mast cell function and DNA damage response Experimental groups: Model+si-NC, Model+ si-PBX3. (A) RT-qPCR and western blot analyses were conducted to evaluate the mRNA expression levels of PBX3 and TOP2A. ∗∗∗ p < 0.001 vs. the Model+si-NC group. (B–D) Western blotting and IF staining were used to assess the expression of the DNA damage marker γ-H2AX. ∗ p < 0.05, ∗∗∗ p < 0.001 vs. the Model+si-NC group; scale bars, 50 μm. (E) SA-β-gal staining was performed to detect cellular senescence. ∗∗ p < 0.01 vs. the Model+si-NC group; scale bars, 50 μm. (F–H) RT-qPCR, western blotting, and IF staining were employed to examine PARP-1 expression in mast cells. ∗∗∗ p < 0.001 vs. the Model+si-NC group; scale bars, 50 μm. (I) Western blot analysis was used to detect the protein levels of Mito-AIF, Cyto-AIF, and Nucleo-AIF. ∗∗∗ p < 0.001 vs. the Model+si-NC group. (J and K) ELISA assays were conducted to quantify the levels of β-hexosaminidase, histamine, IL-4, IL-5, and IFN-γ in mast cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+si-NC group. Data are represented as mean ± SEM ( n = 3 per group) from independent biological replicates. Two-tailed unpaired Student’s t tests for (A, B, D–G, and I–K).

Article Snippet: Cells were then incubated overnight at 4°C with a primary antibody against PARP-1 (PB9309, BOSTER, Pleasanton, CA, USA, 1:100).

Techniques: Cell Function Assay, Quantitative RT-PCR, Western Blot, Expressing, Staining, Marker, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: PBX3 regulates mast cell parthanatos via TOP2A mediated DNA damage in allergic rhinitis

doi: 10.1016/j.isci.2026.115426

Figure Lengend Snippet: PBX3 drives DNA damage through TOP2A to regulate mast cell parthanatos, senescence, and degranulation Experimental groups: Model+si-NC, Model+si-PBX3, Model+si-PBX3+OE-NC, Model+si-PBX3+OE-TOP2A. (A) Western blot analysis was performed to determine protein expression levels of PBX3, TOP2A, PARP-1, and γ-H2AX. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+si-NC or Model+si-PBX3+OE-NC group; ns, not significant ( p ≥ 0.05). (B) IF staining was conducted to detect the DNA damage marker γ-H2AX. ∗∗∗ p < 0.001 vs. the Model+si-NC or Model+si-PBX3+OE-NC group; scale bars, 50 μm. (C) Cellular senescence was assessed using an SA-β-gal staining kit. ∗∗∗ p < 0.001 vs. the Model+si-NC or Model+si-PBX3+OE-NC group; scale bars, 50 μm. (D) ELISA was employed to measure the levels of degranulation markers β-hexosaminidase and histamine. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+si-NC or Model+si-PBX3+OE-NC group. (E) Cytokine levels of IL-4, IL-5, and IFN-γ in cell supernatants were quantified by ELISA. ∗ p < 0.05, ∗∗∗ p < 0.001 vs. the Model+si-NC or Model+si-PBX3+OE-NC group. Data are represented as mean ± SEM ( n = 3 per group) from independent biological replicates. One-way ANOVA with Tukey’s post hoc correction for (A–E).

Article Snippet: Cells were then incubated overnight at 4°C with a primary antibody against PARP-1 (PB9309, BOSTER, Pleasanton, CA, USA, 1:100).

Techniques: Western Blot, Expressing, Staining, Marker, Enzyme-linked Immunosorbent Assay