Journal: Oncotarget

Article Title: Endoplasmic reticulum chaperone prolyl 4-hydroxylase, beta polypeptide (P4HB) promotes malignant phenotypes in glioma via MAPK signaling.

doi: 10.18632/oncotarget.18026

Figure Lengend Snippet: Figure 5: Transient over-expression of P4HB promoted glioma cell proliferation, migration, invasion and tube formation ability in vitro. (A) Western blot analysis showed upregulated expression of P4HB post-transfection. (B) MTT assay was performed on cells with transient P4HB over-expression (D54 P4HB, U87 P4HB and U251 P4HB) and empty vector controls (D54 Vec, U87 Vec and U251 Vec). After 5 days incubation, cells over-expressing P4HB showed higher proliferative rates than controls. (C) Migration assay showed greater motility of U87 P4HB and U251 P4HB than their respective vector controls (U87 Vec and U251 Vec). (D) Matrigel cell invasion assay similarly showed greater invasiveness in U87 and U251 cells with P4HB over-expression (** p <0.05). (E) Angiogenesis, as measured by tube formation ability, was again higher in P4HB overexpressing cells (U87 and U251). (F) Western blot analysis revealed that P4HB over-expression was associated with increased MAPK phosphorylation. (G) Suppression of MAPK activities by U0126 reduced VEGF expression in P4HB over-expressing cells, while P4HB expression level was unaffected. (H) Representative pictures from three independent assays showed decreased cell invasion abilities of U87 P4HB and U251 P4HB cells after treatment with U0126. (I) U0126 pretreatment also inhibited tube formation in these cells after 24 h (Magnification: ×200).

Article Snippet: Briefly, after blocking with 5% non-fat milk in TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6), the membrane was probed with one of the following primary antibodies (at 1:1000 dilution) at 4oC overnight: rabbit monoclonal antibodies against P4HB, total p44/42 MAP kinase (Erk 1/2) (total MAPK), phosphor-p44/42 MAP kinase (Erk1/2) (Thr202/Tyr204) (pMAPK), and VEGF (all from Cell Signaling Technology Inc.).

Techniques: Over Expression, Migration, In Vitro, Western Blot, Expressing, Transfection, MTT Assay, Plasmid Preparation, Incubation, Invasion Assay, Phospho-proteomics

Journal: Translational Oncology

Article Title: The tumor suppressor DACT3 sensitizes triple-negative breast cancer to apatinib by inhibiting the Wnt/β-catenin pathway

doi: 10.1016/j.tranon.2025.102509

Figure Lengend Snippet: DACT3 and apatinib both inhibited β-catenin signalling pathway. (A, B, C, D) Immunofluorescence results of interaction between DACT3 and active catenin, total catenin, VEGFR2, p-VEGFR2. (E, F) Expression of Wnt/β-catenin-related proteins in cells transfected with DACT3 and treated with Apatinib.

Article Snippet: Following overnight incubation using primary antibodies total-β-catenin (#8480; 1:200), non-phospho (active) β-catenin (Ser45) (#19,807; 1:200), phospho-VEGFR2 (Tyr951) (#4991, 1:200, Cell Signaling Technology), and VEGFR2 (#bs-10412R; 1:200, Bioss Antibodies) under 4 °C, DyLight-conjugated anti-rabbit antibody (CoWin Biotech Co., Ltd.) was added for further cell treatment at 37°C for 60 min. And then nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Roche, Palo Alto, CA, USA).

Techniques: Immunofluorescence, Expressing, Transfection

Journal: Cancer Biology & Therapy

Article Title: Lenvatinib potentiates the antitumor efficacy of combined radiotherapy and PD-L1 blockade in lung adenocarcinoma

doi: 10.1080/15384047.2025.2610526

Figure Lengend Snippet: Radiation induces dual adaptive resistance pathways, and lenvatinib counteracts RT-induced PD-L1 upregulation. (A-B) Western blot analysis (A) and quantification (B) of PD-L1 protein expression in HCC827 cells 24 hours after gradient X-ray irradiation (0-20 Gy, n = 3). (C-D) Western blot analysis (C) and quantification (D) of PD-L1 expression in HCC827 cells 24 hours after 8 Gy irradiation, treated with lenvatinib (10 µM) and/or anti-PD-L1 antibody (40 µg/mL, n = 3). (E-F) Representative IF images (E) and quantification (F) of PD-L1 surface expression (MFI) in A549 cells treated with 8 Gy RT and Lenvatinib (10 µM, n = 3). Scale bar = 50 µm. (G) ELISA analysis of secreted VEGF-A protein in the supernatants of HCC827 cells at indicated time points (0-24 h) after 8 Gy irradiation ( n = 3). (H) RT-qPCR analysis of VEGFA mRNA expression in HCC827 cells 24 hours after 8 Gy irradiation ( n = 3). (I-J) Representative IF images (I) and quantification (J) of VEGFR2 expression in HUVECs ( n = 3). Scale bar = 50 µm. Data are presented as mean ± SEM. Statistical analysis was performed using Student's t-test, Mann-Whitney U test, or One-way ANOVA. (ns: P ≥ 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

Article Snippet: Proteins (40 μg) were separated via SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk in TBST (0.05% Tween-20) for one hour at RT, and incubated overnight at 4 °C with primary antibodies: VEGFR2 (D5B1 Rabbit mAb, 1:1,000, CST #9698), PD-L1 (1:1,000, Abcam #ab213524), and GAPDH (D16H11 XP® Rabbit mAb, 1:1,000, CST #5174).

Techniques: Western Blot, Expressing, Irradiation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cancer Biology & Therapy

Article Title: Lenvatinib potentiates the antitumor efficacy of combined radiotherapy and PD-L1 blockade in lung adenocarcinoma

doi: 10.1080/15384047.2025.2610526

Figure Lengend Snippet: Lenvatinib inhibits tumour-induced endothelial activation via VEGFR2 blockade and angiogenic disruption. (A, C) Western blot analysis of VEGFR2 expression in HUVECs treated with increasing concentrations of lenvatinib (0-40 μM) for 24 hours ( n = 3). (B, D) Representative IF images (B) and quantification (D) of VEGFR2 expression (MFI) in HUVECs treated with lenvatinib (10 µM, n = 3). Scale bar = 50 µm. (E) Schematic of the in vitro co-culture model, with HCC827 cells cultured on a 0.4-μm transwell insert above a monolayer of HUVECs (Created with BioRender.com). (F) CCK-8 viability assay of HUVECs cultured alone (monoculture) or co-cultured with HCC827 cells, with or without lenvatinib (10 µM) for 24 hours ( n = 6). (G-I) In vitro tube formation assay using HUVECs cultured in conditioned medium from irradiated (8 Gy) HCC827 cells, which were pretreated with or without lenvatinib. (G) Representative images of Calcein AM-stained HUVEC networks (4-8 h). (H) Quantification of the number of junctions. (I) Quantification of total vascular length ( n = 3). Scale bar = 50 µm. Data are presented as mean ± SEM. Statistics: Mann-Whitney U test or One-way ANOVA. (ns: P ≥ 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

Article Snippet: Proteins (40 μg) were separated via SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk in TBST (0.05% Tween-20) for one hour at RT, and incubated overnight at 4 °C with primary antibodies: VEGFR2 (D5B1 Rabbit mAb, 1:1,000, CST #9698), PD-L1 (1:1,000, Abcam #ab213524), and GAPDH (D16H11 XP® Rabbit mAb, 1:1,000, CST #5174).

Techniques: Activation Assay, Disruption, Western Blot, Expressing, In Vitro, Co-Culture Assay, Cell Culture, CCK-8 Assay, Viability Assay, Tube Formation Assay, Irradiation, Staining, MANN-WHITNEY

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Re regulates PFKFB3-mediated glycolysis to inhibit endothelial cell migration to ameliorate atherosclerosis

doi: 10.1016/j.jgr.2025.11.012

Figure Lengend Snippet: Re suppresses ox-LDL-induced HUVEC proliferation and migration. (A, B) Results of the CCK-8 assay, n = 6. (C) Representative images of HUVECs at 0 h and 12 h after ox-LDL induction in wound healing experiments, bar = 50 μm. (D) Quantification of EC migration in the wound healing assay, n = 3. (E) Western blot assay and quantitative data of VE-cadherin and VEGFR2 in HUVECs, n = 3. ### p < 0.001, ## p < 0.01, # p < 0.05, vs. control group, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, vs. ox-LDL group.

Article Snippet: Ginsenoside Re, (Cat. B21055 , Shanghai Yuanye Biotechnology Co., Ltd, purity≥ 98 %); Simvastatin tablets (Cat. 20210925, Shandong Xinqi Pharmaceutical Co., Ltd); PFKFB3 antibody (Cat. D7H4Q, Cell Signaling Technology, Inc.); HIF-1α Rabbit pAb (Cat. A11945, ABclonal Biotechnology Co., Ltd); HK2 polyclonal antibody (Cat. 22029-1-AP, Wuhan Sanying Biotechnology Co., Ltd); vascular endothelial cadherin (VE-cadherin) antibody (Cat. 2158S, Cell Signaling Technology, Inc.); VEGFA Rabbit mAb (Cat. ab214424, abcam); VEGFR2 Rabbit mAb (Cat. 2479, Cell Signaling Technology, Inc.).

Techniques: Migration, CCK-8 Assay, Wound Healing Assay, Western Blot, Control

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Re regulates PFKFB3-mediated glycolysis to inhibit endothelial cell migration to ameliorate atherosclerosis

doi: 10.1016/j.jgr.2025.11.012

Figure Lengend Snippet: Re inhibits endothelial cell migration via the PFKFB3-HIF-1α/VEGFA-VEGFR2 signaling pathways. (A) Western blot assay and quantitative analysis of PFKFB3 in HUVECs, n = 3. (B) Cell viability of PFKFB3 overexpressing ECs measured by CCK-8 assay, n = 6. (C) Western blot assay and quantitative analysis of PFKFB3 in HUVECs, n = 3. (D–E) Representative images of HUVEC induced by ox-LDL for 0 h and 12 h in wound healing experiments (bar = 50 μm) and quantification of EC migration, n = 3. (F, G) Western blot assay and quantitative data of VE-cadherin, HIF-1α, VEGFA, and VEGFR2 in HUVECs, n = 3. ### p < 0.001, ## p < 0.01, # p < 0.05, vs. control group; ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, vs. ox-LDL group.

Article Snippet: Ginsenoside Re, (Cat. B21055 , Shanghai Yuanye Biotechnology Co., Ltd, purity≥ 98 %); Simvastatin tablets (Cat. 20210925, Shandong Xinqi Pharmaceutical Co., Ltd); PFKFB3 antibody (Cat. D7H4Q, Cell Signaling Technology, Inc.); HIF-1α Rabbit pAb (Cat. A11945, ABclonal Biotechnology Co., Ltd); HK2 polyclonal antibody (Cat. 22029-1-AP, Wuhan Sanying Biotechnology Co., Ltd); vascular endothelial cadherin (VE-cadherin) antibody (Cat. 2158S, Cell Signaling Technology, Inc.); VEGFA Rabbit mAb (Cat. ab214424, abcam); VEGFR2 Rabbit mAb (Cat. 2479, Cell Signaling Technology, Inc.).

Techniques: Migration, Protein-Protein interactions, Western Blot, CCK-8 Assay, Control