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