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: HIPK2-and IKKβ-dependent phosphorylation stabilizes TAp63α during the oocyte DNA damage response

doi: 10.64898/2026.04.17.719163

Figure Lengend Snippet: (A) Schematic overview of the experimental approach used to assess kinase-dependent regulation of TAp63α following DNA damage. (B) Western blot analysis showing a phosphorylation-dependent mobility shift of TAp63α upon doxorubicin treatment. This shift is reduced by CHK2 and CK1 inhibition and is further diminished following calf intestinal phosphatase (CIP) treatment, confirming phosphorylation-dependent modification. (C) Inhibition of HIPK2 or IKKβ, individually or in combination, reduces TAp63α phosphorylation and is associated with decreased protein stability. In contrast, inhibition of CHK2 or CK1 reduces phosphorylation without affecting total TAp63α levels. (D) Schematic representation of the siRNA-mediated knockdown strategy in stable TAp63α-expressing H1299 cells. (E) Knockdown of HIPK2 or IKKβ reduces the phosphorylation-associated mobility shift of TAp63α compared to control and scrambled siRNA conditions. Immunoblotting with phospho-serine/threonine antibodies further confirms a reduction in overall phosphorylation levels upon kinase depletion. (F) Western blot analysis of TAp63α following inhibition of CHK2, HIPK2, and IKKβ under DNA damage conditions. CHK2 inhibition reduces the phosphorylation-associated mobility shift of TAp63α. Inhibition of HIPK2 and IKKβ decreases total TAp63α protein stability. Co-inhibition of CHK2 with HIPK2 and IKKβ restores TAp63α protein levels when applied prior to or concurrently, but not when CHK2 inhibition is applied after HIPK2 and IKKβ inhibition. (G) Schematic representation of the in vivo experimental workflow. (H) Western blot analysis of mouse ovarian lysates showing that cisplatin induces a phosphorylation-associated mobility shift of TAp63α. Co-treatment with HIPK2 or IKKβ inhibitors reduces both phosphorylation and total TAp63α levels. Five pairs of ovaries were pooled per sample. (I) Schematic overview of the experimental approach used for oocyte analysis. (J–K) Representative images showing distinct patterns of TAp63α nuclear distribution in oocytes, along with their quantification. Treatment with HIPK2 or IKKβ inhibitors under DNA damage conditions results in a shift toward reduced nuclear signal intensity. Statistical significance for Class III follicles: doxorubicin + HIPK2 inhibitor (P = 0.0001) and doxorubicin + IKKβ inhibitor (P = 0.0001) compared to doxorubicin alone. Data are presented as mean ± SD; unpaired t-test.

Article Snippet: After 20 minutes, DNA damage was induced using doxorubicin (10 μM; Cat no. 15007, Cayman).

Techniques: Western Blot, Phospho-proteomics, Mobility Shift, Inhibition, Modification, Knockdown, Expressing, Control, In Vivo

Journal: bioRxiv

Article Title: HIPK2-and IKKβ-dependent phosphorylation stabilizes TAp63α during the oocyte DNA damage response

doi: 10.64898/2026.04.17.719163

Figure Lengend Snippet: (A) Schematic overview of the experimental workflow. (B) Western blot analysis showing that inhibition of HIPK2 and IKKβ reduces TAp63α phosphorylation and total protein levels. Treatment with the proteasome inhibitor MG132 restores TAp63α protein levels. (C) Protein interaction network analysis identifying MDM4 as a potential TAp63α-interacting partner. (D–E) Western blot analysis of TAp63α levels following inhibition of MDM4 or MDM2 in the presence of HIPK2 and IKKβ inhibitors. MDM4 inhibition restores TAp63α protein levels, whereas MDM2 inhibition shows a partial effect. (F) Schematic representation of the experimental design for ubiquitination assays. (G–H) Western blot of ubiquitination assays showing increased mono- and poly-ubiquitination of TAp63α upon inhibition of HIPK2 and IKKβ. Co-inhibition of MDM4 reduces TAp63α ubiquitination levels. (I) Schematic overview of the experimental approach for interaction analysis. (J) Co-immunoprecipitation analysis showing interaction between TAp63α and MDM4 under basal and DNA damage conditions. Reduced interaction is observed following DNA damage in wild-type TAp63α, whereas non-phosphorylatable mutants retain interaction with MDM4. (K–L) Western blot of ubiquitination assays comparing wild-type and phosphorylation-deficient TAp63α mutants, showing increased ubiquitination of the mutant proteins. Data are presented as mean ± SD; statistical significance determined by unpaired t-test.

Article Snippet: After 20 minutes, DNA damage was induced using doxorubicin (10 μM; Cat no. 15007, Cayman).

Techniques: Western Blot, Inhibition, Phospho-proteomics, Ubiquitin Proteomics, Immunoprecipitation, Mutagenesis

Journal: bioRxiv

Article Title: HIPK2-and IKKβ-dependent phosphorylation stabilizes TAp63α during the oocyte DNA damage response

doi: 10.64898/2026.04.17.719163

Figure Lengend Snippet: (A) Schematic representation of the experimental design. (B) Representative images of ovarian sections from 9-day-postpartum (dpp) C57BL/6 mice treated with Vehicle control (VC), Cisplatin, Cisplatin + HIPK2 inhibitor, Cisplatin + IKKβ inhibitor, and Cisplatin + HIPK2 & IKKβ inhibitors. Ovaries were immunostained for MVH (green, marking germ cells), TAp63α (red, marking DNA damage response), and DNA (blue, nuclear staining). (C) Quantification of the follicles from panel (B). Statistical analysis shows significant differences in follicle counts for Vehicle Control ( P = 0.0001 ), Cisplatin + HIPK2 inhibitor ( P = 0.0001 ), Cisplatin + IKKβ inhibitor ( P = 0.0013 ), and Cisplatin + combined inhibitors ( P = 0.0001 ) compared to the only Cisplatin group. (D) Schematic representation of the experimental design. Representative images of primordial, primary and secondary follicles from ovarian sections of 9-day-postpartum (dpp) C57BL/6 mice treated with Vehicle control, Cisplatin, Cisplatin + HIPK2 inhibitor, Cisplatin + IKKβ inhibitor. Ovaries were immunostained for gH2AX (brown) (E) and for cleaved caspase 3 (F) (brown) with hematoxylin counterstaining. Quantification of the (G) number of gH2AX foci per Oocyte. Statistical significance in comparison to cisplatin group: Vehicle control ( P = 0.0034 ), Cisplatin + HIPK2 inhibitor ( P = 0.0023 ), Cisplatin + IKKβ inhibitor ( P = 0.0020 ) (H) % of follicles positive for gH2AX. Statistical significance in comparison to cisplatin group: Vehicle control ( P = 0.0018 ), Cisplatin + HIPK2 inhibitor ( P = 0.0037 ), Cisplatin + IKKβ inhibitor ( P = 0.04 ) (I) % of follicles positive for cleaved caspase 3. Statistical significance in comparison to cisplatin group: Vehicle control ( P = 0.0017 ), Cisplatin + HIPK2 inhibitor ( P = 0.0052 ), Cisplatin + IKKβ inhibitor ( P = 0.010 ). unpaired t -test, Data are presented as mean ± SD. Scale bars for 9dpp mice ovary section: 100μm, Primordial and primary follicles:10μm, Secondary follicles:5μm.

Article Snippet: After 20 minutes, DNA damage was induced using doxorubicin (10 μM; Cat no. 15007, Cayman).

Techniques: Control, Staining, Comparison

Journal: bioRxiv

Article Title: HIPK2-and IKKβ-dependent phosphorylation stabilizes TAp63α during the oocyte DNA damage response

doi: 10.64898/2026.04.17.719163

Figure Lengend Snippet: (A) Quantification of primordial, primary, and secondary follicles at 9 days post-partum (dpp) in ovaries from mice treated with Vehicle control, Cisplatin, Cisplatin + HIPK2 inhibitor, Cisplatin + IKKβ inhibitor, and Cisplatin + HIPK2 & IKKβ inhibitors. Statistical analysis difference in primordial follicle counts compared to the cisplatin group with P-values as follows: Vehicle control **** P ≤ 0.0001 , Cis + i_HIPK2 **** P ≤ 0.0001 , Cis + i_IKKβ **** P ≤ 0.0001 , and Cis + i_HIPK2 + i_IKKβ **** P ≤ 0.0001 (B) Schematic representation of the experimental design for long-term ovarian assessment under genotoxic stress. (C) Representative images of ovarian sections from 65dpp C57BL/6 mice treated with Vehicle control, Cisplatin, Cisplatin + HIPK2 inhibitor, Cisplatin + IKKβ inhibitor, Cisplatin +HIPK2+IKKβ inhibitor. Ovarian sections were immunostained for TAp63α (brown) with hematoxylin counterstaining. (D) Quantification of the follicles from panel. Statistical analysis indicates significant difference in Vehicle control ( P ≤ 0.0001 ), Cisplatin + HIPK2 inhibitor ( P ≤ 0.0001 ), Cisplatin + IKKβ inhibitor ( P ≤ 0.0013 ), and Cisplatin + combined inhibitors ( P ≤ 0.0001 ) compared to the Cisplatin-only. (E) Quantification of primordial, primary, and secondary follicles, Statistical analysis of primordial follicle counts shows significant differences with P-values as follows: Vehicle control **** P ≤ 0.0001 , Cis + i_HIPK2 **** P ≤ 0.0001 , Cis + i_IKKβ **** P ≤ 0.0001 , and Cis + i_HIPK2 + i_IKKβ **** P ≤ 0.0001 . Statistical analysis was performed using an unpaired t-test . Data are presented as mean ± SD. Non-significant P-values are not displayed in the figure. Scale bars for 9dpp mice ovary section: 100μm and for 60 days mice ovary section: 50μm.

Article Snippet: After 20 minutes, DNA damage was induced using doxorubicin (10 μM; Cat no. 15007, Cayman).

Techniques: Control

Journal: bioRxiv

Article Title: HIPK2-and IKKβ-dependent phosphorylation stabilizes TAp63α during the oocyte DNA damage response

doi: 10.64898/2026.04.17.719163

Figure Lengend Snippet: (A) Schematic representation of the experimental design for intraperitoneal administration of vehicle control (VC), HIPK2 inhibitor, and IKKβ inhibitor under normal and DNA damage conditions. Following treatment, ovarian tissues were collected for mRNA isolation and subsequent RT-PCR analysis. (B) Quantification of the relative fold change in expression of pro-apoptotic genes ( Puma and Noxa ) and anti-apoptotic genes ( Bcl2l1 and Mcl1 ) across different treatment conditions, including VC, HIPK2 inhibitor, IKKβ inhibitor, cisplatin, cisplatin + HIPK2 inhibitor, and cisplatin + IKKβ inhibitor. Statistical analysis of gene expression changes with unpaired t -tests. The P values are as follows: Puma: VC vs. HIPK2 inhibitor ( P ≤ 0.0016 ), VC vs. IKKβ inhibitor ( P ≤ 0.0045 ), VC vs. cisplatin (ns), cisplatin vs. cisplatin + HIPK2 inhibitor ( P ≤ 0.0235 ), cisplatin vs. cisplatin + IKKβ inhibitor ( P ≤ 0.0236 ). Noxa: VC vs. HIPK2 inhibitor ( P ≤ 0.0181 ), VC vs. IKKβ inhibitor ( P ≤ 0.0188 ), VC vs. cisplatin ( P ≤ 0.035 ), cisplatin vs. cisplatin + HIPK2 inhibitor ( P ≤ 0.0018 ), cisplatin vs. cisplatin + IKKβ inhibitor ( P ≤ 0.0031 ). Bcl2L1: VC vs. HIPK2 inhibitor ( P ≤ 0.0016 ), VC vs. IKKβ inhibitor ( P ≤ 0.0005 ), VC vs. cisplatin ( P ≤ 0.0264 ), cisplatin vs. cisplatin + HIPK2 inhibitor ( P ≤ 0.0024 ), cisplatin vs. cisplatin + IKKβ inhibitor ( P ≤ 0.0067 ). Mcl1: VC vs. HIPK2 inhibitor ( P ≤ 0.0005 ), VC vs. IKKβ inhibitor (P ≤ 0.0044), VC vs. cisplatin ( P ≤ 0.0008 ), cisplatin vs. cisplatin + HIPK2 inhibitor ( P ≤ 0.0004 ), cisplatin vs. cisplatin + IKKβ inhibitor ( P ≤ 0.0023 ). (C) Schematic representation of the experimental workflow. (D) Quantification of the relative fold change in expression of pro-apoptotic genes ( Bbc3 ) and anti-apoptotic genes ( Mcl1 ) across different treatment conditions, including VC, Doxo, Doxo + HIPK2 inhibitor, and Doxo + IKKβ inhibitor. The P values are as follows: BBC3: VC vs. Doxo ( P ≤ 0.017 ), Doxo vs. Doxo + HIPK2 inhibitor ( P ≤ 0.0091 ), Doxo vs. Doxo + IKKβ inhibitor ( P ≤ 0.0021 . MCL1: VC vs. Doxo ( P ≤ 0.0001 ), Doxo vs. Doxo + HIPK2 inhibitor ( P ≤ 0.0044 ), Doxo vs. Doxo + IKKβ inhibitor ( P ≤ 0.0024 ). Statistical analysis was performed using an unpaired t-test . Data are presented as mean ± SD.

Article Snippet: After 20 minutes, DNA damage was induced using doxorubicin (10 μM; Cat no. 15007, Cayman).

Techniques: Control, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Gene Expression