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: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Screening of bottleneck reaction steps in the 1w–SmHBA operon by multienzyme catalysis. a, Schematic illustration of the reaction components. The in vitro reaction system consisted of crude cell extracts (CCE) from strains harboring the 1w–SmHBA plasmid as the basic catalytic source, supplemented with multienzyme-expressing strains carrying plasmids encoding 4–5, 2–3, or single heterologously expressed Cob enzymes to alter the enzyme composition in each reaction. b, Comparison of in vitro HBA production using CCE from strains H1, H2, and LvH0. Two-sided unpaired t -test is carried out between H1, H2, and LvH0. Unpaired t -test of data: H1 to H2, ∗∗∗∗, P < 0.0001 (t = 35.12); LvH0 to H2, ∗∗∗∗, P < 0.0001 (t = 30.84). c, In vitro HBA production using CCE from H21–H28 strains, each harboring an additional RcCob enzyme. Red bars indicate values higher than those of the H2 reactant, whereas blue bars indicate values lower than those of H2 reactant. Two-sided unpaired t -test is carried out between H1 to H21-28. Unpaired t -test of data:H2 to H21, ∗, P = 0.0227 (t = 3.605); H24 to H2, ∗∗∗∗, P < 0.0001 (t = 24.60); H27 to H2, ∗, P = 0.5363 (t = 0.6757). d, In vitro HBA production using CCE from H37–H44 strains, in which SmCob enzymes were replaced with the corresponding RcCob enzymes. Two-sided unpaired t -test is carried out between H1 to H37-44. Unpaired t -test of data:H37 to H2, ∗, P = 0.0145 (t = 4.129); H41 to H2, ∗, P = 0.0315 (t = 3.246); H42 to H2, ∗∗∗, P = 0.0003 (t = 11.66); H43 to H2, ∗, P = 0.0229 (t = 3.592); H44 to H2, ∗∗∗∗, P < 0.0001 (t = 15.76). e, Screening of reactions supplemented with crude cell extracts from strains heterologously expressing 4–5 Cob enzymes. HBA-A: CCE with pET28a–CobAIGJM; HBA-B: CCE with pACYCDuet-1–CobFKLH. Unpaired t -test of data: HBA-A 45 OD 600 to Control in HBA titer, ns, P = 0.0729 (t = 2.418); HBA-B 45 OD 600 to Control in HBA titer, ∗∗, P = 0.0029 (t = 6.502); HBA-A 45 OD 600 to Control in Urogen III titer, ∗∗, P = 0.0022 (t = 7.002); HBA-B 45 OD 600 to Control in Urogen III titer, ∗∗, P = 0.0011 (t = 8.309); f, Screening of reactions supplemented with crude cell extracts from strains heterologously expressing 2–3 Cob enzymes. AIG: CCE with pet28a-CobAIG; JM: CCE with pet28a-CobJM; FK: CCE with pet28a-CobFK; LH: CCE with pet28a-CobLH. Unpaired t -test of data: AIG 30 OD600 to Control in Urogen III titer, ∗∗∗, P = 0.0009 (t = 8.801); JM 30 OD600 to Control in Urogen III titer, ∗∗∗∗, P < 0.0001 (t = 24.39); FK 30 OD600 to Control in Urogen III titer, ∗, P = 0.0304 (t = 3.285); LH 30 OD600 to Control in Urogen III titer, ∗∗∗∗, P < 0.0001 (t = 15.93); g, Screening of reactions supplemented with CCE from strains heterologously expressing a single Cob enzyme. Unpaired t -test of data: CobA + to ori, ∗∗∗, P = 0.0002 (t = 13.95); CobI + to ori, ∗∗∗, P = 0.0005 (t = 10.57); CobG + to ori, ∗∗∗, P = 0.0003 (t = 12.00); CobJ + to ori, ∗∗∗, P = 0.0001 (t = 15.48); CobM + to ori, ∗∗∗, P = 0.0005 (t = 10.46); CobF + to ori, ∗∗∗∗, P < 0.0001 (t = 16.42); CobK + to ori, ∗∗∗, P = 0.0006 (t = 9.873); CobL + to ori, ∗∗, P = 0.0042 (t = 5.882); CobH + to ori, ∗∗∗, P = 0.001 (t = 8.675).

Article Snippet: A representative example is the HBA synthetic pathway: after the porphyrin ring compound Urogen III is assembled, nine successive enzymatic reactions modify the macrocycle by introducing eight methyl groups and removing a methylene bridge, thereby altering its symmetry.

Techniques: In Vitro, Plasmid Preparation, Expressing, Comparison, Control

Journal: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Optimization of the artificial HBA operon. a, In vitro HBA production using CCE from various strains under different modifications. promoter replacement (H2 vs. H46 and H47, trc or tac promoters): Two-sided unpaired t -test is carried out between H2 to H46 and H47. Unpaired t -test of data: H46 to H2, ∗∗∗∗, P < 0.0001 (t = 23.77); H47 to H2, ∗∗∗∗, P < 0.0001 (t = 33.93); chassis variation (H2 vs. H48–H52, different E. coli strains harboring the SmHBA plasmid): Two-sided unpaired t -test is carried out between H2 to H48-52, but no significant differences were observed; culture medium effects (H2 grown in different media): Unpaired t -test of data: 2YT to LB, ∗, P = 0.0168 (t = 3.954); and operon composition (H2 vs. H53 and H54, carrying the FKLHIGJM or FKLH + operon): Two-sided unpaired t -test is carried out between H2 to H53 and H54. Unpaired t -test of data: H53 to H2, ∗∗∗∗, P < 0.0001 (t = 44.32). b, Schematic illustration of the ASmHBA (H2 strain), FKLHIGJM (H53 strain), and FKLH + (H54 strain) operons. c, In vitro HBA synthesis using CCEs from H57 cultivated in 2YT medium, supplemented with gradient amounts of either CCE or CobA enzyme. d. Chromatogram of synthetic HBA in CCE3 group, CCE1 group in (c) with standards. UroIII-STD, uroporphyrinogen III standard; HBA-STD, hydrogenobyrinate acid standard; CCE3 and CCE1, reactants using 1-fold H57 CCE and 3-fold H57 CCE in (c).

Article Snippet: A representative example is the HBA synthetic pathway: after the porphyrin ring compound Urogen III is assembled, nine successive enzymatic reactions modify the macrocycle by introducing eight methyl groups and removing a methylene bridge, thereby altering its symmetry.

Techniques: In Vitro, Plasmid Preparation

Journal: Synthetic and Systems Biotechnology

Article Title: Reconstructing the hydrogenobyrinic acid synthetic toolkit by combining cell-free systems and metabolic engineering

doi: 10.1016/j.synbio.2026.01.012

Figure Lengend Snippet: Optimization of the HBA synthetic system. a, HBA titers obtained from screening single Cob enzyme supplementation in the H53∗ CCE reaction: the control H53 CCE reaction, H53–CobA + (H53 CCE with 4 g/L CobA added), and H53–CCE + (H53 CCE with threefold CCE input). ∗ indicates that H53 CCE was prepared from cultures grown in LB medium, whereas unmarked H53 CCE was prepared from cultures grown in 2YT medium. Statistical Significance without bracket denotes unpaired t -test comparisons between each supplemented group (A+, I+, G+, J+, M+, F+, K+, L+, H+) and the corresponding Ori group under the same CCE introduction condition. Unpaired t -test of data: H53-CobA + to H53∗, ∗∗∗, P = 0.0004 (t = 10.67), H53-CCE + to H53∗, ∗∗∗∗, P < 0.0001 (t = 31.05). b, Urogen III accumulation under the same conditions as in (a). Unpaired t -test of data: H53-CobA + to H53∗, ns, P = 0.2509 (t = 1.341), H53-CCE + to H53∗, ∗∗∗∗, P < 0.0001 (t = 61.82). c, Schematic illustration of HBA biosynthesis with SAM supplementation. Abbreviation: 5-ALA, 5-Aminolevulinate; PBG, porphobilinogen; HMB, hydroxymethylbilane; L-Met, l -methionine; SAM, S-adenosyl- l -methionine; SAH, S-adenosyl- l -homocysteine; SRH, S-ribosyl- l -homocysteine. d, Orthogonal combinations of PpK, MetK, and MtnN enzymes used to enhance HBA production via SAM supplementation. The color intensity of the bar corresponds to the magnitude of the values for enhanced visual clarity. e, HBA titers in the optimized SAM supplementation system by adjusting ATP synthesis through varying AMP and SHMP inputs.

Article Snippet: A representative example is the HBA synthetic pathway: after the porphyrin ring compound Urogen III is assembled, nine successive enzymatic reactions modify the macrocycle by introducing eight methyl groups and removing a methylene bridge, thereby altering its symmetry.

Techniques: Control