luciferase assay  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: IFIT3-DVL interaction promotes malignant progression of lung squamous cell carcinoma and large-cell lung carcinoma via canonical WNT signaling

doi: 10.1016/j.jbc.2026.111368

Figure Lengend Snippet: Effect of IFIT3 on cell malignancy and canonical WNT signaling. A and B , Western blot analysis. GAPDH was an internal control. Quantitative analysis of band intensities is provided in . p-GSK-3β (Ser9), the serine nine phosphorylation of GSK-3β. C–H , quantitative real-time PCR analysis. β-actin was an internal control. Vec, vector, the control group of the IFIT3 expression plasmid. IF3, transfection of IFIT3 expression plasmid. si-IF3, siRNA-IFIT3. si-NC, siRNA-negative control, a scrambled siRNA selected as a negative control. si-IF3 (IF3), following IFIT3 knockdown with siRNA, its expression was restored by transfection with IFIT3 expression plasmid. I–K , dual-luciferase assay. IFIT3 expression plasmid or IFIT3 siRNA was transfected to LUSC/LCLC cells, with or without the treatment of recombinant WNT3A protein. (i) Negative Control: transfection of empty plasmid vector or siRNA-negative control. (ii) Positive Control: treatment of recombinant WNT3A protein. (iii) Test Group 1: transfection of IFIT3 expression plasmid or IFIT3 siRNA. (iv) Test Group 2: transfection of IFIT3 expression plasmid or IFIT3 siRNA, with the treatment of recombinant WNT3A protein. rhW3A, the cells were treated with recombinant human WNT3A protein (100 ng/ml) for 12 h. Error bars indicated mean ± SEM (n = 3). Data were analyzed by two-tailed unpaired Student’s t test for ( C , D , F and G ), and one-way ANOVA with Tukey’s post hoc test for ( E , H–K ). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. LCLC, large-cell lung carcinoma; LUSC, lung squamous cell carcinoma.

Article Snippet: For analysis of WNT3A response, cells were treated with recombinant human WNT3A protein (100 ng/ml, R&D systems, Cat# 5036-WN-500/CF) for 12 h prior to dual-luciferase assay or Western blot analysis.

Techniques: Western Blot, Control, Phospho-proteomics, Real-time Polymerase Chain Reaction, Plasmid Preparation, Expressing, Transfection, Negative Control, Knockdown, Luciferase, Recombinant, Positive Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: IFIT3-DVL interaction promotes malignant progression of lung squamous cell carcinoma and large-cell lung carcinoma via canonical WNT signaling

doi: 10.1016/j.jbc.2026.111368

Figure Lengend Snippet: Effect of IFIT3 on canonical WNT signaling. A , Western blot analysis. GAPDH was an internal control. Quantitative analysis of band intensities is provided in A . Vec, vector, is the control group of the IFIT3 expression plasmid. IF3, transfection of IFIT3 expression plasmid. After transfection with IFIT3 expression plasmid, cells were treated for 24 h with dimethyl sulfoxide (10 μM; IFIT3+ dimethyl sulfoxide group) or XAV-939 (10 μM; IFIT3+XAV-939 group). B , dual-luciferase assay. TOPFLASH/FOPFLASH was used to measure for β-catenin/TCF mediated transcriptional activity. C , nuclear and Cytoplasmic Protein Extraction Assay analysis. Cyto, cytoplasm; Nuc, nucleus; Lamin B1 was a reference nuclear protein, and α-tubulin was a positive control for the cytoplasm. Quantitative analysis of band intensities is provided in B . D , immunofluorescence analysis (the scale bar represents 10 μm). Quantification of average fluorescence intensity was provided. E , representative immunohistochemistry images for IFIT3 and β-catenin in LUSC and LCLC (magnification: × 400; the scale bar represents 50 μm). F , Kaplan-Meier survival curve analyzing the different group in cancerous samples from LUSC/LCLC patients. a, the group with cytoplasmic IFIT3 and nuclear β-catenin coexpression (16 cases); b, the group with IFIT3 and β-catenin coexpression in the cytoplasm (56 cases); c. others (110 cases). Log-rank test was used for survival comparison. Error bars indicated mean ± SEM (n = 3). Data were analyzed by one-way ANOVA with Tukey’s post hoc test for ( B ), and two-tailed unpaired Student’s t test for ( D ). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. LCLC, large-cell lung carcinoma; LUSC, lung squamous cell carcinoma.

Article Snippet: For analysis of WNT3A response, cells were treated with recombinant human WNT3A protein (100 ng/ml, R&D systems, Cat# 5036-WN-500/CF) for 12 h prior to dual-luciferase assay or Western blot analysis.

Techniques: Western Blot, Control, Plasmid Preparation, Expressing, Transfection, Luciferase, Activity Assay, Protein Extraction, Positive Control, Immunofluorescence, Fluorescence, Immunohistochemistry, Comparison, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: IFIT3-DVL interaction promotes malignant progression of lung squamous cell carcinoma and large-cell lung carcinoma via canonical WNT signaling

doi: 10.1016/j.jbc.2026.111368

Figure Lengend Snippet: IFIT3 interacts with DVL2 to promote DVL2 phosphorylation and upregulate β-catenin expression. A , co-immunoprecipitation analysis. IP, immunoprecipitation. IB, immunoblot. B , immunofluorescence assay (the scale bar represents 50 μm). Quantitative colocalization analysis of these images is presented in . C, E and F , Western blot analysis. GAPDH was an internal control. Quantitative analysis of band intensities is provided in . p-GSK-3β (Ser9), the serine nine phosphorylation of GSK-3β. p-β-catenin (Ser675), the serine 675 phosphorylation of β-catenin. ABC, active non-phospho (Ser33/37/Thr41) β-catenin. p-DVL2 (Ser143), the serine 143 phosphorylation of DVL2. p-DVL2 (Thr224), the threonine 224 phosphorylation of DVL2. D , dual-luciferase assay. TOPFLASH/FOPFLASH was measured for β-catenin/TCF mediated transcriptional activity. Cells were co-transfected with siRNA and plasmid. Vec, vector, the control group of the IFIT3 expression plasmid. IF3, transfection of IFIT3 expression plasmid. si-D1, siRNA-DVL1. si-D2, siRNA-DVL2. si-D3, siRNA-DVL3. si-NC, siRNA-negative control, a scrambled siRNA selected as a negative control. si-IF3 (IF3), following IFIT3 knockdown with siRNA, its expression was restored by transfection with IFIT3 expression plasmid. Data were analyzed by one-way ANOVA with Tukey’s post hoc test for ( D ). Error bars indicated mean ± SEM (n = 3). ∗ p < 0.05.

Article Snippet: For analysis of WNT3A response, cells were treated with recombinant human WNT3A protein (100 ng/ml, R&D systems, Cat# 5036-WN-500/CF) for 12 h prior to dual-luciferase assay or Western blot analysis.

Techniques: Phospho-proteomics, Expressing, Immunoprecipitation, Western Blot, Immunofluorescence, Control, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Negative Control, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: IFIT3-DVL interaction promotes malignant progression of lung squamous cell carcinoma and large-cell lung carcinoma via canonical WNT signaling

doi: 10.1016/j.jbc.2026.111368

Figure Lengend Snippet: Effect of IFIT3 on cell malignancy and canonical WNT signaling. A and B , Western blot analysis. GAPDH was an internal control. Quantitative analysis of band intensities is provided in . p-GSK-3β (Ser9), the serine nine phosphorylation of GSK-3β. C–H , quantitative real-time PCR analysis. β-actin was an internal control. Vec, vector, the control group of the IFIT3 expression plasmid. IF3, transfection of IFIT3 expression plasmid. si-IF3, siRNA-IFIT3. si-NC, siRNA-negative control, a scrambled siRNA selected as a negative control. si-IF3 (IF3), following IFIT3 knockdown with siRNA, its expression was restored by transfection with IFIT3 expression plasmid. I–K , dual-luciferase assay. IFIT3 expression plasmid or IFIT3 siRNA was transfected to LUSC/LCLC cells, with or without the treatment of recombinant WNT3A protein. (i) Negative Control: transfection of empty plasmid vector or siRNA-negative control. (ii) Positive Control: treatment of recombinant WNT3A protein. (iii) Test Group 1: transfection of IFIT3 expression plasmid or IFIT3 siRNA. (iv) Test Group 2: transfection of IFIT3 expression plasmid or IFIT3 siRNA, with the treatment of recombinant WNT3A protein. rhW3A, the cells were treated with recombinant human WNT3A protein (100 ng/ml) for 12 h. Error bars indicated mean ± SEM (n = 3). Data were analyzed by two-tailed unpaired Student’s t test for ( C , D , F and G ), and one-way ANOVA with Tukey’s post hoc test for ( E , H–K ). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. LCLC, large-cell lung carcinoma; LUSC, lung squamous cell carcinoma.

Article Snippet: For analysis of WNT3A response, cells were treated with recombinant human WNT3A protein (100 ng/ml, R&D systems, Cat# 5036-WN-500/CF) for 12 h prior to dual-luciferase assay or Western blot analysis.

Techniques: Western Blot, Control, Phospho-proteomics, Real-time Polymerase Chain Reaction, Plasmid Preparation, Expressing, Transfection, Negative Control, Knockdown, Luciferase, Recombinant, Positive Control, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Quantitative high-throughput screening identifies small-molecule modulators of Wnt/β-catenin signaling

doi: 10.3389/fphar.2026.1788041

Figure Lengend Snippet: β-lactamase activity concentration-response to compounds in LEF/TCF-bla FreeStyle™ 293F cell in 1536-well format. (A) Stimulation concentration response curve of human WNT-3A (hWNT-3A). (B) Stimulation concentration response curves of the Wnt potentiator R-Spondin2 (hRS) in combination with different concentrations of hWNT-3A (hRS alone, 75, 150, and 300 ng/mL). (C) Curves showing the mild effect of imatinib mesylate on cell viability and its inhibitory effect on stimulator-induced β-lactamase activity, confirming assay responsiveness to known Wnt pathway antagonists.

Article Snippet: Recombinant human WNT-3A (hWNT-3A) and R-spondin 2 (hRS) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activity Assay, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Quantitative high-throughput screening identifies small-molecule modulators of Wnt/β-catenin signaling

doi: 10.3389/fphar.2026.1788041

Figure Lengend Snippet: Western blot confirmation of identified inhibitors. (A) Representative Western blot analysis showing the effects of each compound on β-catenin protein levels. Treatment with the stimulator (DMSO +) markedly increased both non-phosphorylated (active) and total β-catenin compared with the DMSO control (DMSO -). All known inhibitors attenuated the stimulator-induced increases. (B) Quantitative densitometry of non-phosphorylated and total β-catenin from three independent experiments, normalized to the housekeeping protein GAPDH. Data are presented as mean ± SD. Statistical significances relative to the DMSO + control is indicated above bars (*p < 0.05; **p < 0.01; ***p < 0.001). “+” denotes the addition of stimulator; “-” denotes its absence. The stimulator consisted of 75 ng/mL hWNT-3A and 95 ng/mL hRS.

Article Snippet: Recombinant human WNT-3A (hWNT-3A) and R-spondin 2 (hRS) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control