Journal: Nature Communications

Article Title: Gadd45g insufficiency drives the pathogenesis of myeloproliferative neoplasms

doi: 10.1038/s41467-024-47297-2

Figure Lengend Snippet: a c-kit + BM cells from diseased Gadd45g +/ − and Ctrl mice were lysed, precipitated with anti-GADD45g antibody, and detected by Western blot with anti-RAC2 and -GADD45g antibodies. b Representative immunofluorescence micrographs showing colocalization of GADD45g with RAC2 in c-kit + BM cells of Ctrl mice. Panels represent nucleus (blue), GADD45g (green), RAC2 (yellow), and merged images, respectively. Arrows in merged image indicate colocalization of GADD45g with RAC2. Bar represents 10 μm. c Representative immunofluorescence micrographs showing cellular distribution of GADD45g, RAC2 and RAC1 in cord blood CD34 + cells from healthy human donors. Panels represent nucleus (blue), GADD45g (green), RAC2 (orange), RAC1 (pink), and merged images, respectively. Arrows in merged image indicate colocalization of GADD45g with RAC2. Bar represents 5 μm. d Western blot analysis of RAC2-GTP and total RAC2 protein levels in c-kit + BM cells from diseased Gadd45g +/− and Ctrl mice. e HEL and SET-2 cells transfected with GADD45g-specific shRNA or shCtrl were lysed, precipitated with anti-GADD45g antibody, and detected by Western blot with anti-RAC2 and -GADD45g antibodies. f Western blot analysis of RAC2-GTP and total RAC2 protein levels in HEL and SET-2 cells transfected with GADD45g- specific shRNA or shCtrl. g Western blot analysis of p-PAK1 and total PAK1 protein levels in c-kit + BM cells from diseased Gadd45g +/ − and Ctrl mice. h Western blot analysis of p-PAK1 and total PAK1 protein levels in HEL and SET-2 cells transfected with GADD45g- specific shRNA or shCtrl. i HEL and SET-2 cells were transfected with GADD45g- specific shRNA or shCtrl for 48 h, followed by transfection with RAC2-specific shRNA (shRAC2) or scrambled control (Scr) for another 48 h. The protein levels of p-PAK1, total PAK1, p-PI3K, total PI3K, pAKT-Ser473 and total AKT were examined by Western blot. j HEL and SET-2 cells were transfected with GADD45g- specific shRNA or shCtrl for 48 h, followed by treatment with vehicle or IPA-3 (10 μM for 48 h). The protein levels of p-PI3K, total PI3K, pAKT-Ser473 and total AKT were examined by Western blot. For ( a–j ): At least three independent experiments with similar results were performed.

Article Snippet: AKT inhibitor MK-2206 2HCI, PAK1 inhibitor IPA-3, RAC inhibitor EHT 1864 2HCI, JAK2 inhibitor ruxolitinib, and HDAC1/2 inhibitor Romidepsin were all purchased from SelleckChem (Houston, Texas, USA).

Techniques: Western Blot, Immunofluorescence, Transfection, shRNA, Control

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 expression is adversely associated with the overall survival of ovarian cancer patients. (A) Genetic alterations of PAK1 across different cancer types, as retrieved from the cBioPortal database. (B) PAK1 expression patterns across a variety of cancers. (C) Association between PAK1 expression levels and copy number variations. (D) Correlation of PAK1 expression with homologous recombination (HR) status. (E) Kaplan–Meier survival analysis comparing ovarian cancer patient outcomes with low ( n = 467) and high ( n = 188) PAK1 expression levels.

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: Expressing, Homologous Recombination

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: Nature Communications

Article Title: Gadd45g insufficiency drives the pathogenesis of myeloproliferative neoplasms

doi: 10.1038/s41467-024-47297-2

Figure Lengend Snippet: a qRT-PCR analysis of GADD45g expression in BMMNCs from patients with PV treated with 50 nM ruxolitinib (Rux) or vehicle for 24 h ( n = 5). b Western blot analysis of JAK2, p-JAK2 and GADD45g protein levels in 293T cells transduced with lentiviruses expressing JAK2V617F , wild-type JAK2 or empty vector constructs. Blots shown are representative of three independent experiments. c HEL92.1.7-Luc cells were transfected with Dox-inducible GADD45g -specific shRNA and engrafted into irradiated NSG mice. Bioluminescence imaging of representative mice was taken at day 10 post-transplantation ( n = 3 mice per group). d The NSG mice were treated with either vehicle or Rux for 2 weeks ( n = 20 mice per group). Bioluminescence imaging of representative mice from each group were taken ( n = 3 mice per group). e qRT-PCR evaluation of GADD45g expression in hCD45-positive cells isolated from the BMMNCs of mice in each group ( n = 3 mice per group). f , g Bioluminescence imaging of representative mice from the indicated groups were taken after 1 week of shRNA expression induced by Dox treatment ( f ). Kaplan-Meier survival curves of mice in each group were shown ( n = 8–9 mice per group, log-rank test) ( g ). h–j MPN cell lines and BMMNCs from MPNs patients were treated with 2 μM Decitabine (DAC) for 96 h. qRT-PCR analysis of GADD45g expression in MPN cell lines ( h ) or BMMNCs from MPNs patients ( n = 3 patients) ( i ). MS-PCR showing the methylated GADD45g alleles in MPN cell lines (M, methylated allele; U, unmethylated allele). Bands shown are representative of three independent experiments ( j ). k , l Correlations of the expression between GADD45g and HDAC1 ( k ) or HDAC2 ( l ) in CD34 + BMMCs/PBMCs from MPNs patients. ( m ) qRT-PCR evaluation of GADD45g expression in MPN cell lines treated with 3 nM Romidepsin (Romi) or DMSO for 48 h. n qRT-PCR analysis of GADD45g expression in BMMNCs from MPNs patients treated with 5 nM Romi or DMSO for 48 h ( n = 3). o , p HEL ( o ) and SET-2 cells ( p ) were treated with 3 nM Romi or DMSO for 48 h, and then subjected to ChIP analysis. The enriched DNA which associated with the promoter region of GADD45g was quantified by qPCR. For ( a , e , h , i , k , p ): Data are shown as means ± SD, two-tailed Student’s t test. For ( h , m , o , p ) : Figures shown are representative of three independent experiments with similar results. n = 3 technical replicates.

Article Snippet: AKT inhibitor MK-2206 2HCI, PAK1 inhibitor IPA-3, RAC inhibitor EHT 1864 2HCI, JAK2 inhibitor ruxolitinib, and HDAC1/2 inhibitor Romidepsin were all purchased from SelleckChem (Houston, Texas, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transduction, Plasmid Preparation, Construct, Transfection, shRNA, Irradiation, Imaging, Transplantation Assay, Isolation, Methylation, Two Tailed Test