Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: PAK1 regulates homologous recombination (HR) repair and olaparib sensitivity in ovarian cancer cells. (A, B) HR (A) and non-homologous end-joining (NHEJ) (B) repair efficiencies in control and PAK1-depleted HEK293T cells, evaluated using HR and NHEJ reporter systems. Data were presented as mean ± standard error of the mean from three independent experiments. (C) Western blotting analysis of PAK2 and PAK3 in control and PAK2/PAK3-depleted HEK293T cells. (D – G) HR (D, F) and NHEJ (E, G) repair efficiencies in control and PAK2/PAK3-depleted HEK293T cells, evaluated using HR and NHEJ reporter systems. (H, I) Cell cycle distribution in control and PAK1-depleted Ovcar8 cells, analyzed by flow cytometry. (J, K) RAD51 foci formation in Ovcar8 cells treated with 10 μM olaparib for 24 h: (J) representative images and (K) quantification. More than 200 cells were analyzed per experiment. (L, N) The survival of control or PAK1-depleted Ovcar8 (L) and SKOV-3 (N) cells, assessed by colony formation assay. (M, O) Phosphorylation of CHK1 in control or PAK1-depleted Ovcar8 (M) and SKOV-3 (O) cells, treated with 10 μM olaparib for 6 h. (P, Q) The survival of control, (P) PAK2-depleted, or (Q) PAK3-depleted Ovcar8 cells, assessed by colony formation assay. Error bars represent the standard error of the mean from three independent experiments. Scale bars = 50 μm.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Homologous Recombination, Non-Homologous End Joining, Control, Western Blot, Flow Cytometry, Colony Assay, Phospho-proteomics

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: PAK1 regulates homologous recombination (HR) repair and olaparib sensitivity dependent on its kinase activity. PAK1-depleted cells were transfected with wild-type PAK1 or the K299R kinase mutant for 24 h. (A) Western blotting analysis of PAK1 and CHK1 phosphorylation in transfected Ovcar8 cells treated with 10 μM olaparib for 6 h. (B) The survival of transfected Ovcar8 cells treated with different concentrations of olaparib for 2 weeks, assessed by colony formation assay. (C) HR activity in transfected HEK293T cells co-transfected with HR reporter plasmids, followed by HR assay after 48 h. (D, E) RAD51 foci formation in transfected Ovcar8 cells treated with 10 μM olaparib for 24 h: (D) representative images and (E) quantification. Over 200 cells were analyzed in each experiment. Error bars represent the standard error of the mean from three independent experiments.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Homologous Recombination, Activity Assay, Transfection, Mutagenesis, Western Blot, Phospho-proteomics, Colony Assay

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: PAK1 inhibition enhances the efficiency of olaparib in ovarian cancer cells. (A) Western blotting analysis of PAK1 and CHK1 phosphorylation in Ovcar8 cells treated with 10 μM olaparib, 10 μM IPA-3, or their combination for 6 h. (B) Homologous recombination (HR) efficiency in HEK293T cells transfected with HR reporter plasmids, treated with olaparib, IPA-3, or both, followed by HR assay. (C, D) RAD51 foci formation in Ovcar8 cells treated with olaparib, IPA-3, or their combination for 24 h: (C) representative images and (D) quantification. More than 200 cells were analyzed per experiment. (E, F) The survival of Ovcar8 (E) and SKOV-3 (F) cells treated with olaparib alone or in combination with IPA-3, assessed by colony formation assay. Error bars represent the standard error of the mean from three independent experiments. Statistical significance was determined by a two-tailed t -test, with P -values < 0.05 considered significant.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Inhibition, Western Blot, Phospho-proteomics, Homologous Recombination, Transfection, Colony Assay, Two Tailed Test

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: PAK1 inhibition promotes olaparib-induced replication stress and DNA damage. (A, B) DNA fiber assay for the length of CIdU (red) tracks in Ovcar8 cells treated with olaparib, IPA-3, or their combination for 6 h: (A) representative images and (B) quantification. Data were expressed as mean ± standard deviation, analyzed by a two-tailed unpaired t -test. (C, D) Immunoblot analysis of chromatin and soluble fractions of Ovcar8 cells treated with olaparib, IPA-3, or both for 6 h, probing for the indicated antibodies (C), or IPOND (isolation of proteins on nascent DNA) analysis of RPA1 and RPA2 at replication forks (D). (E, F) γ-H2AX foci formation in Ovcar8 cells treated with olaparib, IPA-3, or their combination for 24 h: (E) representative images and (F) quantification. More than 100 cells were counted per experiment. (G) RNA sequencing analysis of Ovcar8 cells treated with olaparib and IPA-3. Differentially expressed genes were classified based on fold change ≥ 1.5 or ≤ 0.5, with P < 0.05. (H) Biological process analysis of up- and down-regulated genes in the combination treatment compared with IPA-3 alone. (I, J) GSEA of up- and down-regulated genes in the combination treatment compared with IPA-3 alone. (K) The heatmap displaying up-regulated genes associated with DNA repair. (L) The quantitative real-time PCR showed the up-regulated genes associated with DNA repair. Error bars represent the standard error of the mean from three independent experiments. Statistical significance was determined by a two-tailed t -test. Two-sided P -values < 0.05 were considered significant. Scale bars = 50 μm.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Inhibition, Standard Deviation, Two Tailed Test, Western Blot, Isolation, RNA Sequencing, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: Combination of IPA-3 and olaparib synergistically suppresses ovarian cancer xenograft tumor growth. OVCAR8 and SKOV-3 cells were subcutaneously implanted into NOD-SCID mice, and the animals were treated with control (DMSO), IPA-3 (10 mg/kg), olaparib (50 mg/kg), or their combination (intraperitoneally, 3 days × 6 times). (A, B, L, M) Serum AST and ALT were measured for Ovcar8 (A, B) and SKOV-3 (L, M) xenografts. (C, D, N, O) Tumor images and growth curves for Ovcar8 (C, D) and SKOV-3 (N, O) xenografts. Data were expressed as mean ± standard error of the mean from five independent samples. Statistical significance was assessed by a two-tailed unpaired t -test. (E–K, P – V ) Hematoxylin-eosin, Ki-67, γ-H2AX, and cleaved caspase-3 staining in tumor tissues, evaluated by immunohistochemistry for Ovcar8 (E–K) and SKOV-3 (P–V) xenografts. Quantification is shown in the corresponding panels. Images of 10 random fields per section were analyzed using ImageJ software. Scale bars = 50 μm. Statistical analysis was performed using a two-tailed t -test and two-way ANOVA. P -values < 0.05 were considered significant.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Control, Two Tailed Test, Staining, Immunohistochemistry, Software

Journal: Genes & Diseases

Article Title: PAK1 inhibition synergistically enhances the anti-tumor efficacy of PARP inhibitors in ovarian cancers

doi: 10.1016/j.gendis.2025.101887

Figure Lengend Snippet: Combination of IPA-3 and olaparib synergistically suppresses ovarian cancer cells' growth in patient-derived organoid and patient-derived xenograft models. (A, B, D, E) Ovarian cancer organoids were treated with IPA-3 (200 nM), olaparib (200 nM), or both for 3 days. Representative bright-field images and quantitative analysis are shown. (C, F) Western blotting analysis of CHK1 phosphorylation and cleaved caspase-3 in ovarian cancer organoids treated with IPA-3 (200 nM), olaparib (200 nM), or both for 3 days. (G, H) Patient-derived xenograft models were established by transplanting tumor tissues into 6-week-old female BALB/c nude mice. Mice were treated with DMSO, IPA-3 (10 mg/kg), olaparib (50 mg/kg), or their combination. Tumor images (G) and growth curves (H) are shown. (I–O) Immunohistochemical analysis of hematoxylin-eosin, cleaved caspase-3, γ-H2AX, and Ki-67 levels in tumor tissues. Quantification of staining is shown in (K), (M), and (O). Data were represented as mean ± standard deviation. Images of 10 random fields per section were recorded for analysis. Statistical significance was assessed using a two-tailed t -test and a two-way ANOVA. P -values < 0.05 were considered significant. Scale bars = 50 μm.

Article Snippet: Chlorodeoxyuridine (Cidu), 5-iodo-2′-deoxyuridine (Idu), and the PAK1 inhibitor IPA-3 were sourced from MedChemExpress, while the PARP inhibitor olaparib was acquired from TargetMol.

Techniques: Derivative Assay, Western Blot, Phospho-proteomics, Immunohistochemical staining, Staining, Standard Deviation, Two Tailed Test

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: