ipa 3  (MedChemExpress)

Journal: Clinical and Translational Medicine

Article Title: ATF6‐mediated GTPase RHOQ promotes liver cancer progression via enhancing aerobic glycolysis through PAK1 phosphorylation

doi: 10.1002/ctm2.70871

Figure Lengend Snippet: RHOQ interacts with PAK1 and promotes its phosphorylation. (A) Immunoprecipitation assay for the binding of HA‐PAK1 to flag‐RHOQ, flag‐RHOQ(Q67L) and flag‐RHOQ(T23N) in Huh7 cells. Immunoprecipitation was performed using anti‐HA and anti‐flag antibodies, and immunoblotting analysis was performed using PAK1 and RHOQ antibodies. (B) Immunoprecipitation assay for the binding of flag‐RHOQ to HA‐PAK1 and HA‐del‐PAK1 in Huh7 cells. (C) Immunoblotting analysis for PAK1 and del‐PAK1 phosphorylation levels in Huh7 cells. (D) Molecular docking simulates the binding of RHOQ to PAK1; green represents RHOQ, and blue represents PAK1. Yellow dashed lines represent hydrogen bonds. HDOCK web server: http://hdock.phys.hust.edu.cn/ . (E) Immunoblotting analysis for the PAK1 downstream effectors and their phosphorylation in Huh7 cells after IPA‐3 (10 µM) treatment. (F) Immunoblotting analysis for RHOQ downstream effectors and their phosphorylation in Huh7 cells. IPA‐3 concentration was 10 µM. (G) Immunoblotting analysis for RHOQ downstream effectors and their phosphorylation in Huh7 cells. FTY720 concentration was 10 µM. (H) Immunoblotting analysis for ATF6 downstream effectors and their phosphorylation in Huh7 cells. (I) Significantly enriched pathways ( Q < .05) are shown. The x‐axis indicates the Gene Ratio, while the y ‐axis shows the pathway categories. Bubble size reflects the gene count in each pathway, and bubble colour denotes the padj (FDR‐adjusted p value). (J) Represented images for transwell assay of Huh7 cells. IPA‐3 concentration was 10 µM. (K) Represented images for transwell assay of Huh7 cells. FTY720 concentration was 10 µM.

Article Snippet: Huh7 cells were seeded at 3 × 10 4 /well in 24‐well Seahorse XF microplates, transfected with plasmids and treated with 10 μm IPA‐3 for 24 h (MCE, USA).

Techniques: Phospho-proteomics, Immunoprecipitation, Binding Assay, Western Blot, Analysis, Docking Assay, Concentration Assay, Transwell Assay

Journal: Clinical and Translational Medicine

Article Title: ATF6‐mediated GTPase RHOQ promotes liver cancer progression via enhancing aerobic glycolysis through PAK1 phosphorylation

doi: 10.1002/ctm2.70871

Figure Lengend Snippet: RHOQ enhances aerobic glycolysis in liver tumour cells. (A and B). Glycolytic stress assay for ECAR levels in Huh7 cells. IPA‐3 concentration was 10 µM. (C and D) Mitochondrial stress assay for OCR levels in Huh7 cells. IPA‐3 concentration was 10 µM. (E) Detection of extracellular lactate levels in Huh7 cells. Results are presented as mean ± SD ( n = 3; biological replicates). (F) Detection of ATP production in Huh7 cells. Results are presented as mean ± SD ( n = 3; biological replicates). (G) Rows represent individual metabolites and columns represent individual samples. Red and blue indicate significantly up‐ and down‐regulated metabolites, respectively, with colour intensity reflecting the magnitude of log 2 ‐transformed fold change (Log2FC). Hierarchical clustering dendrograms on the left illustrate similarities among metabolites. (H) The pie chart displays the percentage distribution of all differential metabolites categorised by their annotated KEGG pathways. The area of each slice is proportional to the number of differential metabolites in that pathway relative to the total number of differential metabolites. (I and J) The y ‐axis represents the fold change. The boxes represent the interquartile range (IQR) with the median indicated by the internal horizontal line; whiskers extend to the minimum and maximum values. (K) Glycolytic stress assay for ECAR levels in Huh7 cells. (L) Mitochondrial stress assay for OCR levels in Huh7 cells. (M) Represented images for transwell assay of Huh7 cells. (E, F, I and J) By Student's t ‐test. * p < .05, ** p < .01, *** p < .001. Abbreviations : ECAR, extracellular acidification rate; OCR, oxygen consumption rate; G6P, glucose 6‐phosphate; F1,6BP, fructose 1,6‐bisphosphate.

Article Snippet: Huh7 cells were seeded at 3 × 10 4 /well in 24‐well Seahorse XF microplates, transfected with plasmids and treated with 10 μm IPA‐3 for 24 h (MCE, USA).

Techniques: Concentration Assay, Transformation Assay, Transwell Assay

Journal: Clinical and Translational Medicine

Article Title: ATF6‐mediated GTPase RHOQ promotes liver cancer progression via enhancing aerobic glycolysis through PAK1 phosphorylation

doi: 10.1002/ctm2.70871

Figure Lengend Snippet: IPA‐3 synergises with GPC3–CAR T‐cell therapy against HCC. (A) A schematic diagram of GPC3–CAR structure. (B) Flow cytometry analysis for the positive rate of GPC3–CAR transduced T cells. (C) Lactate dehydrogenase (LDH) release assay for the killing activity of GPC3–CAR T cells against Huh7 target cells in different treatment groups. IPA‐3 treatment conditions were 10 µM, 24 h and effector‐to‐target ratios (E/T) were 10:1, 5:1, 2.5:1 and 1.25:1. Results are presented as mean ± SD ( n = 4; biological replicates). (D) Enzyme linked immunosorbent assay for the expression of IFN‐γ and TNF‐α in the supernatant of GPC3–CAR T cells co‐cultured with Huh7 cells in different treatment groups. IPA‐3 treatment conditions were 10 µM, 24 h. Results are presented as mean ± SD ( n = 3; biological replicates). (E) Experimental mode of combined use of GPC3–CAR T cells and IPA‐3. Cell and drug injection dosage and frequency were described in the Material and Methods section. (F) Macroscopic images of tumours after GPC3–CAR T cells, IPA‐3 or combination therapy ( n = 6; biological replicates). (G and H) Quantitative data of tumour weight (G) and volume (H) after GPC3–CAR T cells, IPA‐3 or combination therapy. Results are presented as mean ± SD ( n = 6; biological replicates). (I) CFSE assay for T cell proliferation, IPA‐3 treatment at 10 µM for 24 h. (J and K) Flow cytometry analysis for changes in T cell effector (J) and exhaustion (K) markers, IPA‐3 treatment at 10 µM for 24 h. (C) By two‐way ANOVA; (D, G and H) by one‐way ANOVA. * p < .05, ** p < .01, *** p < .001, ns, not significant. Abbreviation : E/T ratio, effector/target cell ratio.

Article Snippet: Huh7 cells were seeded at 3 × 10 4 /well in 24‐well Seahorse XF microplates, transfected with plasmids and treated with 10 μm IPA‐3 for 24 h (MCE, USA).

Techniques: Flow Cytometry, Analysis, Lactate Dehydrogenase Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Injection, CFSE 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 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