Journal: Molecular Biomedicine

Article Title: Microwave dynamic therapy induces ferroptosis in colorectal cancer by targeting PTK2B to regulate STAT3-mediated GPX4 expression

doi: 10.1186/s43556-025-00322-2

Figure Lengend Snippet: MWDT modulates GPX4 expression via PTK2B to regulate ferroptosis. a Relative mRNA expression levels of PTK2B in HCT15 and HCT116 cells treated with MWDT, with and without PTK2B inhibition. b Western blotting analysis of PTK2B levels in HCT15, and HCT116 cells receiving different treatments. c Immunofluorescence assay showed that the expression of PTK2B was decreased in the MWDT group compared with the empty vector group. d Western blotting analysis of PTK2B, P-PTK2B, and GPX4 levels in HCT15, and HCT116 cells receiving different treatments. e The relative levels of GSH were measured in HCT15 and HCT116 cells treated with MWDT and PTK2B inhibition. f – h Flow cytometry analysis of ROS levels indicated a marked increase in ROS in both HCT15 and HCT116 cells treated with MWDT, which was reversed by PTK2B inhibition. i - k Flow cytometry analysis of C11-BODIPY levels indicated a marked increase in LPO in both HCT15 and HCT116 cells treated with MWDT, which was reversed by PTK2B inhibition. l MDA levels were significantly elevated in MWDT-treated cells, with a marked decrease observed in the PTK2B inhibition group. m , n Representative colony formation images ( m ) and quantification ( n ) of HCT15 and HCT116 cells show that PTK2B inhibition mitigates the colony formation ability of CRC cells treated with MWDT. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: After blocking in 5% skim milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies against PTK2B (1:1000, Proteintech), ACSL4 (1:1000, Proteintech), GPX4 (1:1000, Proteintech), and ACTIN (1:1000, Proteintech).

Techniques: Expressing, Inhibition, Western Blot, Immunofluorescence, Plasmid Preparation, Flow Cytometry

Journal: Molecular Biomedicine

Article Title: Microwave dynamic therapy induces ferroptosis in colorectal cancer by targeting PTK2B to regulate STAT3-mediated GPX4 expression

doi: 10.1186/s43556-025-00322-2

Figure Lengend Snippet: PTK2B regulates GPX4 transcription through p-STAT3 (Tyr705). a , b PTK2B positively regulated the phosphorylation of STAT3 at Tyr705 in HCT15, and HCT116 cells. c The mRNA expression of GPX4 in HCT15 PTK2B overexpression cell lines, with or without treatment with Stattic (5 µM). d Protein levels of PTK2B, p-PTK2B, STAT3, p-STAT3, and GPX4 in HCT15 PTK2B overexpression cell lines, with or without treatment with Stattic (5 µM). e , f Western blotting and qRT-PCR analyses of the change in GPX4 expression caused by STAT3 overexpression in CRC cells. g JASPAR predicted a conserved STAT3 binding motif, and a schematic diagram of the potential STAT3 binding site in the GPX4 promoter is shown in the figure. h ChIP analysis of STAT3 occupancy at the GPX4 promoter in HCT15 cells and HCT116 cells. i Luciferase reporter assays were conducted in HCT15 and HCT116 cells overexpressing STAT3 and GPX4 reporter plasmids. j - k ChIP-qPCR analysis was performed using anti-STAT3 antibody or control IgG in HCT15 cells with stable PTK2B knockdown or PTK2B overexpression.** p < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: After blocking in 5% skim milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies against PTK2B (1:1000, Proteintech), ACSL4 (1:1000, Proteintech), GPX4 (1:1000, Proteintech), and ACTIN (1:1000, Proteintech).

Techniques: Phospho-proteomics, Expressing, Over Expression, Western Blot, Quantitative RT-PCR, Binding Assay, Luciferase, ChIP-qPCR, Control, Knockdown

Journal: Molecular Biomedicine

Article Title: Microwave dynamic therapy induces ferroptosis in colorectal cancer by targeting PTK2B to regulate STAT3-mediated GPX4 expression

doi: 10.1186/s43556-025-00322-2

Figure Lengend Snippet: p-PTK2B(Tyr402) interacts with STAT3 and promotes nuclear accumulation of p-STAT3 (Tyr705). a Molecular docking analysis showing a strong binding effect between PTK2B and STAT3. b - d Co-IP assays showing the interaction between PTK2B and STAT3 in HCT116, HCT15, and HCT116 PTK2B overexpression cells. Blots were probed for PTK2Band STAT3. e IF staining showing co-localization of STAT3 (red) and PTK2B (green) in HCT116 and HCT15 cells. Nuclei are stained with DAPI (blue). f IF analysis showing nuclear accumulation of p-STAT3 (Tyr705) in HCT116 cells overexpressing PTK2B or vector. p-STAT3 (Tyr705) is shown in green and DAPI in blue. g Western blot analysis showing that overexpression of wild-type PTK2B significantly enhances phosphorylation of STAT3 at tyrosine 705, whereas kinase-inactive PTK2B (K457R) does not. h Co-IP of STAT3-Myc, followed by phosphorylation detection of STAT3 at Tyr705, showing that wild-type PTK2B promotes STAT3 phosphorylation. i In vitro kinase assay showing that GST-PTK2B kinase domain (GST-PTK2B-KD) efficiently phosphorylates STAT3 at Tyr705, while the K457R mutant does not

Article Snippet: After blocking in 5% skim milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies against PTK2B (1:1000, Proteintech), ACSL4 (1:1000, Proteintech), GPX4 (1:1000, Proteintech), and ACTIN (1:1000, Proteintech).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Over Expression, Staining, Plasmid Preparation, Western Blot, Phospho-proteomics, In Vitro, Kinase Assay, Mutagenesis

Journal: Molecular Biomedicine

Article Title: Microwave dynamic therapy induces ferroptosis in colorectal cancer by targeting PTK2B to regulate STAT3-mediated GPX4 expression

doi: 10.1186/s43556-025-00322-2

Figure Lengend Snippet: MWDT regulates the transcription of GPX4 by modulating the PTK2B-STAT3 axis. a MWDT treatment significantly decreased the phosphorylation of STAT3 at Tyr705 in HCT15 and HCT116 cells, without altering total STAT3 protein levels. b Relative mRNA expression levels of STAT3 in HCT15 and HCT116 cells treated with MWDT. c , d Immunofluorescence staining reveals a significant reduction in nuclear p-STAT3 accumulation following MWDT treatment in HCT15 cells and HCT116 cells. e , f Western blot analysis of p-STAT3 and total STAT3 protein levels in HCT15 cells and HCT116 cells treated with MWDT, with or without PTK2B overexpression. g , h ChIP-qPCR results showing reduced binding of STAT3 to the GPX4 promoter in MWDT-treated cells, an effect partially alleviated by PTK2B overexpression. i Immunohistochemical staining for PTK2B, P-STAT3, and GPX4 in tumors. Scale bar: 50 μm

Article Snippet: After blocking in 5% skim milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies against PTK2B (1:1000, Proteintech), ACSL4 (1:1000, Proteintech), GPX4 (1:1000, Proteintech), and ACTIN (1:1000, Proteintech).

Techniques: Phospho-proteomics, Expressing, Immunofluorescence, Staining, Western Blot, Over Expression, ChIP-qPCR, Binding Assay, Immunohistochemical staining

Journal: Molecular Biomedicine

Article Title: Microwave dynamic therapy induces ferroptosis in colorectal cancer by targeting PTK2B to regulate STAT3-mediated GPX4 expression

doi: 10.1186/s43556-025-00322-2

Figure Lengend Snippet: Schematic representation of the MWDT-PTK2B-STAT3-GPX4 axis that synchronically regulates CRC progression. Created with BioRender

Article Snippet: After blocking in 5% skim milk in TBST, membranes were incubated overnight at 4 °C with primary antibodies against PTK2B (1:1000, Proteintech), ACSL4 (1:1000, Proteintech), GPX4 (1:1000, Proteintech), and ACTIN (1:1000, Proteintech).

Techniques:

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 1. Adenoviral-mediated overexpression of CTGF in the chamber angle of the mouse eye. A: Light microscopy: eyes treated with control virus (Ad5-GFP) or Ad5-CTGF show no structural abnormalities in the ciliary body (CB), iris (Ir), TM (arrows), and Schlemm’s canal (SC, asterisk). B: GFP labeling in TM and corneal endothelium (arrows) after Ad5-GFP injection. C: Real-time RT-PCR for CTGF mRNA in limbal tissues of Ad5-GFP– (Co) and Ad5-CTGF–treated eyes 1 to 24 days after treatment. Mean value of Ad5-GFP–treated eyes was set at 1 (*P 0.05, **P 0.01; reference gene: GNB2L). D: Western blot analysis for fibronectin of CTGF in limbal tissues of eyes 2 months after injection of Ad5-GFP or Ad5-CTGF. Coomassie blue staining to confirm equal loading of proteins. E: Immunohistochemistry (IHC) for CTGF (red) in the anterior eye of animals 2 months after Ad5-GFP or Ad5-CTGF treatment. GFP labeling is seen in virus-transduced cells (green). In both experimental and control eyes, immunostaining for CTGF is seen in the corneal endothelium and in the stroma of the ciliary processes. In control eyes, no staining for CTGF is seen in the TM (arrows), whereas the TM of experimental eyes shows distinct immunoreactivity for CTGF (arrows) F: IHC for fibronectin (red) in the chamber angle of eyes 2 months after Ad5-GFP or Ad5-CTGF treatment. GFP labeling in virus-transduced cells (green). In control eyes, fibronectin is seen adjacent to the SC and in the stroma of the ciliary processes. After Ad5-CTGF injection, immunoreactivity for fibronectin around SC is more intense and detectable in the Ir root throughout the CB and the sclera close to the outer wall of SC. G: IHC for -SMA (red) in eyes 2 months after treatment with Ad5-GFP or Ad5-CTGF. In Ad5-GFP–injected eyes, -SMA is detectable in several cells in the TM and around ciliary process vessels. In contrast, in the experimental eye -SMA staining is seen in cells throughout the entire TM and in the stroma of the CB and Ir. Scale bar 50 m.

Article Snippet: Western blot analysis was performed with specific antibodies as described previously.47 Antibodies were used as follows: mouse anti– -SMA (1:500; Serotec), goat anti– -actinin, goat anti-CTGF, rabbit anti-(p-Tyr-397) focal adhesion kinase (all 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-(p-)extracellular signal–regulated kinase 1/2, rabbit anti-integrin 1 (both 1:1000; Cell Signaling Technologies, Beverly, MA), rabbit anti-fibronectin 1:1000 (Dako); mouse anti-vinculin (1:1000; Sigma-Aldrich), chicken anti-goat, chicken anti-mouse, and chicken anti-rabbit IgG, coupled to horseradish peroxidase (all 1:2000; Santa Cruz).

Techniques: Over Expression, Light Microscopy, Control, Virus, Labeling, Injection, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemistry, Immunostaining

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 3. CTGF induces contraction and formation of actin stress fibers in HTM cells. A: Collagen contraction assay with HTM cells seeded in a matrix of collagen type I and treated with CTGF or TGF-2. After 24 hours, culture wells were photographed (bottom) and area of matrices measured (top). The area obtained in controls was set at 1 (n 4; *P 0.05). B: Phalloidin labeling (red) of actin stress fibers in control HTM cells or 24 hours after treatment with CTGF. Nuclear DNA was stained with DAPI (blue). Scale bar 50 m. C: Western blot analysis for -SMA or -actinin in proteins of cultured HTM cells after treatment with 5 and 50 ng/mL of CTGF for 2 hours. Coomassie blue staining to confirm equal loading of proteins. D: Length of phalloidin-labeled stress fibers 24 hours after treatment with CTGF (n 4; *P 0.05). E: Phalloidin labeling (red) of actin stress fibers in HTM cells or 24 hours after treatment with control virus (Ad5-GFP) or Ad5-CTGF. GFP labeling (green) shows virus-infected cells; nuclear DNA is labeled with DAPI (blue). Scale bar 50 m.

Article Snippet: Western blot analysis was performed with specific antibodies as described previously.47 Antibodies were used as follows: mouse anti– -SMA (1:500; Serotec), goat anti– -actinin, goat anti-CTGF, rabbit anti-(p-Tyr-397) focal adhesion kinase (all 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-(p-)extracellular signal–regulated kinase 1/2, rabbit anti-integrin 1 (both 1:1000; Cell Signaling Technologies, Beverly, MA), rabbit anti-fibronectin 1:1000 (Dako); mouse anti-vinculin (1:1000; Sigma-Aldrich), chicken anti-goat, chicken anti-mouse, and chicken anti-rabbit IgG, coupled to horseradish peroxidase (all 1:2000; Santa Cruz).

Techniques: Contraction Assay, Labeling, Control, Staining, Western Blot, Cell Culture, Virus, Infection

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 5. Depletion of CTGF in HTM cells by RNA interference attenuates the actin cytoskeleton. A: Real-time RT-PCR analysis for CTGF mRNA in HTM-N cells or HTM-pSiCTGF cells with stable depletion of CTGF. Mean value of HTM-N cells was set at 1 (**P 0.02, reference gene: GNB2L). B: Western blot analysis for CTGF or fibronectin in HTM-N or pSiCTGF cells; Coomassie blue staining was used to confirm equal loading of proteins. IHC for vinculin (green) and phalloidin labeling (red) of HTM-N or pSiCTGF cells 24 hours after seeding (C) or in confluent areas (D). In confluent areas, HTM-N cells show numerous thick and longitudinally arranged actin stress fibers that are associated with vinculin-labeled focal contacts. In marked contrast, in pSiCTGF cells longitudinally arranged actin stress fibers are barely detectable. After treatment of pSiCTGF cells with 50 ng/mL of CTGF for 12 hours, number and thickness of longitudinally arranged actin stress fibers significantly increases. E: Western blot analysis for -SMA, -actinin, or profilin-1 in HTM-N or pSiCTGF cells after serum starvation for 24 hours. F: Real-time RT-PCR analysis for mRNA of -SMA or profilin-1 in HTM-N or pSiCTGF cells after 24 hours. Mean value of HTM-N cells was set at 1 (**P 0.02; reference gene: GNB2L). G: Western blot analysis for RhoA/GTP and total RhoA, pMLCs and total MLCs, or pFAK and total FAK in HTM-N or pSiCTGF cells after serum free incubation for 3 hours. Coomassie blue staining was used to confirm equal loading of proteins.

Article Snippet: Western blot analysis was performed with specific antibodies as described previously.47 Antibodies were used as follows: mouse anti– -SMA (1:500; Serotec), goat anti– -actinin, goat anti-CTGF, rabbit anti-(p-Tyr-397) focal adhesion kinase (all 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-(p-)extracellular signal–regulated kinase 1/2, rabbit anti-integrin 1 (both 1:1000; Cell Signaling Technologies, Beverly, MA), rabbit anti-fibronectin 1:1000 (Dako); mouse anti-vinculin (1:1000; Sigma-Aldrich), chicken anti-goat, chicken anti-mouse, and chicken anti-rabbit IgG, coupled to horseradish peroxidase (all 1:2000; Santa Cruz).

Techniques: Quantitative RT-PCR, Western Blot, Staining, Labeling, Incubation

Journal: The American journal of pathology

Article Title: Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

doi: 10.1016/j.ajpath.2012.02.030

Figure Lengend Snippet: Figure 6. Analysis of CTGF overexpressing transgenic mice. A: Northern blot analysis for CTGF mRNA in transgenic and WT lenses and whole eyes without lenses. Methylene blue staining used to confirm equal loading. B: Western blot analysis for CTGF in aqueous humor of 1-month-old WT and transgenic B1-CTGF1 mice. C: Light microscopy. In WT and transgenic B1-CTGF1 mice, the iridocorneal angle is wide open, and no structural abnormalities are present in ciliary body (CB), iris (Ir), TM (arrow), and Schlemm’s canal (SC, asterisks). D: IHC for CTGF in the anterior eye of 2-month-old WT and transgenic B1-CTGF1 mice. In the WT animal, immunoreactivity for CTGF in the TM is weak or absent (arrows). In contrast, in the transgenic B1-CTGF1 mouse immunoreactivity for CTGF is seen in the TM (arrows), in the corneal endothelium, and along the inner surface of the cornea. E: IHC for fibronectin in the iridocorneal angle of 2-month-old WT and transgenic B1-CTGF1 mice. In WT animals, fibronectin is seen adjacent to the SC and close to the capillaries in the Ir and CB. In contrast, in transgenic B1-CTGF1 mice, immunoreactivity for fibronectin around the SC and in the stroma of the CB is more intense. F: IHC for -SMA in the iridocorneal angle of 2-month-old WT and transgenic B1-CTGF1 mice. In transgenic B1-CTGF1 mice, intense immunoreactivity for -SMA is present in the TM and the stroma of the CB in contrast to the WT littermate. G: IOP measurement of 1- to 3-month-old WT and transgenic B1-CTGF1 mice. Medians (red) with maximum and minimum (lines) and upper and lower quartile (n 15, *P 0.05). H: Optic nerve sections of 3-month-old WT and transgenic B1-CTGF1 mice showing increased areas devoid of axons in transgenic B1-CTGF1 animals (left; arrows). Number of optic nerve axons in 1- to 3-month-old WT and transgenic B1-CTGF1 mice (right; n 5; **P 0.01). Scale bars: 50 m (C–F); 20 m (H).

Article Snippet: Western blot analysis was performed with specific antibodies as described previously.47 Antibodies were used as follows: mouse anti– -SMA (1:500; Serotec), goat anti– -actinin, goat anti-CTGF, rabbit anti-(p-Tyr-397) focal adhesion kinase (all 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-(p-)extracellular signal–regulated kinase 1/2, rabbit anti-integrin 1 (both 1:1000; Cell Signaling Technologies, Beverly, MA), rabbit anti-fibronectin 1:1000 (Dako); mouse anti-vinculin (1:1000; Sigma-Aldrich), chicken anti-goat, chicken anti-mouse, and chicken anti-rabbit IgG, coupled to horseradish peroxidase (all 1:2000; Santa Cruz).

Techniques: Transgenic Assay, Northern Blot, Staining, Western Blot, Light Microscopy