Journal: PLOS One

Article Title: Strategic selection of MDM2 inhibitors enhances the efficacy of FAK inhibition in mesothelioma based on TP53 genotype

doi: 10.1371/journal.pone.0343551

Figure Lengend Snippet: (A) Growth inhibitory effects of defactinib on mesothelioma and Met-5A cells. Cell cells were treated with defactnib as indicated for 72 hrs and the viability was assayed with the WST assay. IC 50 values and SE bars (n = 3) are also included. (B) Expression level of the molecules associated with FAK and p53 in mesothelioma cells treated with defactinib as indicated for 24 hrs. The western blot analysis included expression levels of a phosphorylated form of FAK, p53 and AKT, and a cleaved form of caspase-9 and PARP. Tubulin was used as a loading control. The expression of each molecule was quantified with ImageJ software (NIH, Bethesda, MD, USA) with tubulin intensity as a normalized control (see ).

Article Snippet: The protein was transferred to a nylon filter membrane and was hybridized with antibody against phosphorylated p53 at Ser 15 (#9284), AKT (#9272), phosphorylated AKT at Ser 473 (#9271), caspase-9 (which also detected cleaved caspase-9) (#9505), PARP (poly ADP-ribose polymerase) (which also detected cleaved PARP) (#4108), FAK (#3285), phosphorylated FAK at Tyr 397 (#3283), phosphorylated MDM2 at Ser 166 (#3521), MERLIN (D1D8, #6995), actin (#4970) (Cell Signaling, Danvers, MA, USA), phosphorylated H2AX (phosphor-2A histone family member X) at Ser 139 (#613401) (BioLegend, San Diego, CA, USA), MDM2 (#413) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Ab-6, clone DO-1, #MS-187-P0), and tubulin-α (DM1A, Thermo Fisher Scientific) followed by an appropriate second antibody ( ).

Techniques: WST Assay, Expressing, Western Blot, Control, Software

Journal: PLOS One

Article Title: Strategic selection of MDM2 inhibitors enhances the efficacy of FAK inhibition in mesothelioma based on TP53 genotype

doi: 10.1371/journal.pone.0343551

Figure Lengend Snippet: (A) Growth inhibitory effects of nutlin-3a on mesothelioma and Met-5A cells. Cell cells were treated with nutlin-3a as indicated for 72 hrs and the viability was assayed with the WST assay. IC 50 values and SE bars (n = 3) are also included. (B) Expression level of the molecules associated with FAK and p53 in mesothelioma cells treated with nultin-3a as indicated for 24 hrs. The western blot analysis also included expression levels of a phosphorylated form of FAK, p53 and H2AX. Actin was used as a loading control. For phosphorylated H2AX, the blots were exposed for a longer period to ensure the detection of basal signals in untreated cells. The basal intensity therefore differed from that of the RITA experiments in where shorter exposures were used. The expression of each molecule was quantified with ImageJ software (NIH, Bethesda, MD, USA) with actin intensity as a normalized control (see ).

Article Snippet: The protein was transferred to a nylon filter membrane and was hybridized with antibody against phosphorylated p53 at Ser 15 (#9284), AKT (#9272), phosphorylated AKT at Ser 473 (#9271), caspase-9 (which also detected cleaved caspase-9) (#9505), PARP (poly ADP-ribose polymerase) (which also detected cleaved PARP) (#4108), FAK (#3285), phosphorylated FAK at Tyr 397 (#3283), phosphorylated MDM2 at Ser 166 (#3521), MERLIN (D1D8, #6995), actin (#4970) (Cell Signaling, Danvers, MA, USA), phosphorylated H2AX (phosphor-2A histone family member X) at Ser 139 (#613401) (BioLegend, San Diego, CA, USA), MDM2 (#413) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Ab-6, clone DO-1, #MS-187-P0), and tubulin-α (DM1A, Thermo Fisher Scientific) followed by an appropriate second antibody ( ).

Techniques: WST Assay, Expressing, Western Blot, Control, Software

Journal: PLOS One

Article Title: Strategic selection of MDM2 inhibitors enhances the efficacy of FAK inhibition in mesothelioma based on TP53 genotype

doi: 10.1371/journal.pone.0343551

Figure Lengend Snippet: (A) Growth inhibitory effects of RITA on mesothelioma and Met-5A cells. Cell cells were treated with RITA as indicated for 72 hrs and the viability was assayed with the WST assay. IC 50 values and SE bars (n = 3) are also included. (B) Expression level of the molecules associated with FAK and p53 in mesothelioma cells treated with RITA as indicated for 24 hrs. The western blot analysis included expression levels of a phosphorylated form of FAK, p53 and H2AX. Actin was used as a loading control. For phosphorylated H2AX of MSTO-211H cells, the blot was exposed for a longer period to ensure the detection of basal signals in untreated cells. The expression of each molecule was quantified with ImageJ software (NIH, Bethesda, MD, USA) with actin intensity as a normalized control (see ).

Article Snippet: The protein was transferred to a nylon filter membrane and was hybridized with antibody against phosphorylated p53 at Ser 15 (#9284), AKT (#9272), phosphorylated AKT at Ser 473 (#9271), caspase-9 (which also detected cleaved caspase-9) (#9505), PARP (poly ADP-ribose polymerase) (which also detected cleaved PARP) (#4108), FAK (#3285), phosphorylated FAK at Tyr 397 (#3283), phosphorylated MDM2 at Ser 166 (#3521), MERLIN (D1D8, #6995), actin (#4970) (Cell Signaling, Danvers, MA, USA), phosphorylated H2AX (phosphor-2A histone family member X) at Ser 139 (#613401) (BioLegend, San Diego, CA, USA), MDM2 (#413) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Ab-6, clone DO-1, #MS-187-P0), and tubulin-α (DM1A, Thermo Fisher Scientific) followed by an appropriate second antibody ( ).

Techniques: WST Assay, Expressing, Western Blot, Control, Software

Journal: PLOS One

Article Title: Strategic selection of MDM2 inhibitors enhances the efficacy of FAK inhibition in mesothelioma based on TP53 genotype

doi: 10.1371/journal.pone.0343551

Figure Lengend Snippet: Cells were treated as follows for 24 hrs and the cell lysate were subjected to western blot analysis, which included expression levels of a phosphorylated form of FAK, p53 and AKT, and a cleaved form of PARP. We selected concentrations lower than the IC 50 values of each single agent to detect the molecular changes induced by the combination. Concentrations of defactinib, nutrin-3a and RITA used were as follows. NCI-H28: 2 μM defactinib, 4 μM nutlin-3a, 0.04 μM RITA; MSTO-211H: 2 μM defactinib, 1 μM nutlin-3a, 1 μM RITA; NCI-H226: 2 μM defactinib, 2 μM nutlin-3a, 0.02 μM RITA; EHMES-1: 2 μM defactinib, 0.25 μM RITA; JMN-1B: 2 μM defactinib, 1.5 μM RITA. Actin was used as a loading control. The expression of each molecule was quantified with ImageJ software (NIH, Bethesda, MD, USA) with actin intensity as a normalized control (see ).

Article Snippet: The protein was transferred to a nylon filter membrane and was hybridized with antibody against phosphorylated p53 at Ser 15 (#9284), AKT (#9272), phosphorylated AKT at Ser 473 (#9271), caspase-9 (which also detected cleaved caspase-9) (#9505), PARP (poly ADP-ribose polymerase) (which also detected cleaved PARP) (#4108), FAK (#3285), phosphorylated FAK at Tyr 397 (#3283), phosphorylated MDM2 at Ser 166 (#3521), MERLIN (D1D8, #6995), actin (#4970) (Cell Signaling, Danvers, MA, USA), phosphorylated H2AX (phosphor-2A histone family member X) at Ser 139 (#613401) (BioLegend, San Diego, CA, USA), MDM2 (#413) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (Ab-6, clone DO-1, #MS-187-P0), and tubulin-α (DM1A, Thermo Fisher Scientific) followed by an appropriate second antibody ( ).

Techniques: Western Blot, Expressing, Control, Software

Journal: Regenerative Biomaterials

Article Title: Magnetic chitosan nanoparticle-exosome hydrogel enhances bladder function in diabetic bladder dysfunction via activating the FAK-p38 MAPK-GATA4 axis in adipose-derived mesenchymal stromal cells

doi: 10.1093/rb/rbag007

Figure Lengend Snippet: Establishing the role of the FAK-p38 MAPK-GATA4 axis in the activation of ADSCs by DLSW. ( A and B ) The levels of p-FAK in the ADSCs of the control group and the DLSW group were investigated by immunofluorescence staining and Western blot. ( C and D ) The expressions of p-p38 MAPK in the ADSCs of the control group, the DLSW group and the DLSW+FAK(−) group were examined by immunofluorescence staining and Western blot. ( E and F ) GATA4 expressions in the ADSCs of the control group, the DLSW group and the DLSW+p38(−) group were assessed by immunofluorescence staining and Western blot. ( G ) Quantifications of VEGF and NGF levels secreted by the cells using ELISA. ( H ) Representative photographs of endothelial-like tube formation in the control group, DLSW group and DLSW+GATA4(−) group (left panel). Differences in the number of endotubes are presented as fold change. Rat MPG were cultured in the respective culture medium (right panel). The longest neurite in each group was identified (indicated by a white arrow). To enhance clarity, the neurite outgrowth was delineated by dashed lines. Quantification was conducted by measuring the neurite growth areas in each treatment group. ( I ) Proposed mechanism of the involvement of the FAK-p38 MAPK-GATA4 axis in the activation of ADSCs by DLSW. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The ADSCs in the control group and DLSW group were incubated with the primary antibody of phosphorylated-FAK (p-FAK) (Santa Cruz Biotechnology, USA) overnight at 4°C and were then exposed to secondary antibody for 30 min.

Techniques: Activation Assay, Control, Immunofluorescence, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: bioRxiv

Article Title: Antimetastatic Sulfonate-Functionalized Mesoporous Silica Nanoparticles Enhance Irinotecan Stability and Delivery for Colorectal Cancer Treatment

doi: 10.1101/2025.11.25.690433

Figure Lengend Snippet: Mechanistic evaluation of (SO 3 - )-MSN-PEG/TA on metastasis, focal adhesion turnover, angiogenesis, and tumor targeting. HCT-116 cells were treated with 200 μg/mL of (SO 3 - )-MSN-PEG/TA for 24 hours. (a) Western blot analysis of p-FAK expression levels. (b) Quantitative analysis of p-FAK protein expression relative to the control group (***p < 0.001, n = 3). (c) Immunofluorescence imaging of paxillin (green) and nuclei (blue). Scale bar: 20 μm. (d) Quantitative comparison of the number of FA per cell between control and treated groups (***p < 0.001, n = 5). (e) Schematic representation of the chicken embryo CAM assay. HCT-116 cells were seeded onto the CAM, and (SO 3 - )-MSN-PEG/TA (1 mg/egg) was administered intravenously on embryonic day 13. (f) Representative photographs of CAM vasculature on day 15. (g) Statistical analysis of vascular density performed using ImageJ software (***p < 0.001, n =5). (h) Schematic illustration of the in vivo tumor-targeting study. HCT-116 tumor-bearing mice were intravenously injected with RITC-labeled (SO3⁻)-MSN-PEG/TA (200 mg/kg) and analyzed by multiphoton microscopy. (i) Visualization of the EPR effect. Blood vessels were labeled with FITC–dextran (green), and RITC-labeled (SO3⁻)-MSN-PEG/TA (red) signals were observed 24 hours post-injection. Scale bar = 100 μm. (j) Schematic of the orthotopic colorectal cancer metastasis model. Luciferase-expressing HCT-116 tumor tissue (2 x 10⁶ cells) was implanted into the cecum wall of NOD-SCID mice to establish an orthotopic colorectal cancer model. Mice were intravenously treated with (SO 3 - )-MSN-PEG/TA (equivalent to NPs dose corresponding to 40 mg/kg of IRI@(SO 3 - )-MSN-PEG/TA). (k) Monitoring of orthotopic tumor growth by IVIS imaging from day 3 to day 31. (l) Ex vivo imaging of metastatic lesions: Post-mortem imaging of mice revealed extensive metastasis, with tumor sites identified in organs such as the liver, spleen, kidneys, stomach, and cecum (indicated by cyan arrows).

Article Snippet: Antibodies against phosphorylated FAK (p-FAK) and paxillin were obtained from Cell Signaling Technology, while GAPDH and secondary IgG antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Imaging, Comparison, Chick Chorioallantoic Membrane Assay, Software, In Vivo, Injection, Labeling, Microscopy, Luciferase, Ex Vivo

Journal: Nutrients

Article Title: Bioactive Phenolics from Vinegar–Egg Accelerates Acute Wound Healing by Activation of Focal Adhesion and Mitogen-Activated Protein Kinase Signaling

doi: 10.3390/nu17162584

Figure Lengend Snippet: Involvement of the FAK pathway in MMP2 and MAPK expression and promotion of cell migration. MEF Cells were seeded in 6-well plates (1.5 × 10 5 cells/well) and treated for 24 h with DMSO (0.1%, v / v ), ( A ) 4-hydroxy-benzoic acid ( 1 ) or ( B ) vanillic acid ( 2 ) at 50 and 100 μM for 24 h. The protein levels of phosphorylated and total FAK and p38 MAPK (p-FAK/FAK and p-p38/p38 MAPK) were analyzed by Western blotting. To confirm equivalent protein amounts, β-actin was employed as a reference protein. All values are expressed as mean ± SEM ( n = 3; * p < 0.05, ** p < 0.01 compared with the DMSO control group).

Article Snippet: Primary antibodies against p-p38 MAPK(#4511), MMP2 (matrix metallopeptidase 2, #13132), and p38 MAPK (#9212) were supplied by Cell Signaling Technology (CST, Danvers, MA, USA), whereas antibodies against β-actin (#sc-47778), phosphorylated focal adhesion kinase (p-FAK, #sc-81493), and focal adhesion kinase (FAK, #sc-1688) were supplied by Santa Cruz Biotechnology (SCB, Dallas, TX, USA).

Techniques: Expressing, Migration, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Fibrosis factor CTGF facilitates VCAM‑1‑dependent monocyte adhesion to osteoarthritis synovial fibroblasts via the FAK and JNK pathways

doi: 10.3892/mmr.2025.13489

Figure Lengend Snippet: FAK activation is involved in CTGF-induced VCAM-1 synthesis and monocyte adhesion. (A and B) VCAM-1 expression (n=3) and (C) THP-1 adhesion (n=4) in OASFs incubated with a FAK inhibitor (10 µM) and then stimulated with CTGF for 24 h. The densitometry analysis of (B) was quantified (n=3; right panels). (D) Western blot analysis showing FAK phosphorylation in OASFs treated with CTGF. The densitometry analysis of (D) was quantified (n=3; lower panels). *P<0.05 vs. control group. # P<0.05 vs. CTGF-treated group. FAK, focal adhesion kinase; CTGF, connective tissue growth factor; VCAM-1, vascular cell adhesion molecule-1; p-, phosphorylated.

Article Snippet: CTGF recombinant protein was obtained from PeproTech, Inc. Antibodies against phosphorylated (p-)FAK (cat. no. 3283S) and p-JNK (cat. no. 9251S) were acquired from Cell Signaling Technology, Inc. Antibodies against VCAM-1 (cat. no. GTX110684), FAK (cat. no. GTX50489), JNK (cat. no. GTX52360) and β-actin (cat. no. GTX109639) were acquired from GeneTex International Corporation.

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Phospho-proteomics, Control