Journal: Nutrients

Article Title: Vitamin C Activates Osteoblastogenesis and Inhibits Osteoclastogenesis via Wnt/β-Catenin/ATF4 Signaling Pathways

doi: 10.3390/nu11030506

Figure Lengend Snippet: Expression of both osteoblast- and osteoclast-regulated proteins in vitamin C-treated rat tibias. ( A ) Western blot image of Wnt3a, β-catenin, and ATF4 and quantitative assay of Wnt3a, β-catenin, and ATF4 protein expression in vitamin C-treated rat tibias. ( B ) Western blot image of p-AKT, p-ERK, p-p38, and p-JNK and quantitative assay of p-AKT, p-ERK, p-p38, and p-JNK protein expression in vitamin C-treated rat tibias. Expression was quantified using ImageJ software relative to that of β-actin. Values represent the mean ± standard deviation. Values with different letters were significantly different according to Duncan’s multiple range test ( P < 0.05).

Article Snippet: Membranes were blocked with 5% bovine serum albumin prior to incubation with specific primary antibodies against BMP-2, RUNX2, Wnt3a, osteocalcin, COL-1 (Abcam, Cambridge, UK), SMAD1/5/8 (Santa Cruz Biotechnology, Dallas, TX, USA), ATF4 (Boster, Pleasanton, CA, USA), osteoprotegerin (OPG), RANK, RANKL (Bioss Antibodies, Woburn, MA, USA), TRAP, cathepsin K (GeneTex, Irvine, CA, USA), β-catenin, phosphorylated serine/threonine kinase (p-AKT), phosphorylated extracellular signal-regulated kinase (p-ERK), p-p38, phosphorylated c-Jun N-terminal kinase (p-JNK), and β-actin (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Software, Standard Deviation

Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) Predicted binding affinity scores of BT against pattern recognition receptors using the DeepAVC model. (B) SPR sensorgrams of BT binding to recombinant human cGAS at the indicated concentrations (0.196-50 µM). (C) Dose–response curve fitted from SPR data in (B). (D) RT-qPCR of IFNB1 mRNA in wild-type (WT), cGAS knockout and STING knockout HT1080 cells treated with BT (50, 75 or 100 µM) or DMSO for 12 h. (E) Secreted type I IFN measured by ELISA in the same cell lines and treatments as in (D). (F) Flow cytometry of VSV-GFP infection in WT, cGAS knockout and STING knockout HT1080 cells treated with BT (50, 75, 100 µM) and infected (MOI = 0.1) for 12 h. (G) Western blots of WT, cGAS knockout and STING knockout HT1080 cells treated as in (D). (H-I) In vivo VSV and HSV-1 challenges in cGAS knockout mice. Left panel is survival curves of mice, right panel is viruses RNA levels in liver measured by RT-qPCR. (J) Confocal micrographs of RAW 264.7 cells treated with DMSO or 50 µM BT for 12 h, stained for DAPI (blue) and cytosolic dsDNA (green). Scale bars, 5 µm. (K) Confocal images of RAW 264.7 cells treated as in (J), stained with DAPI (blue) and MitoTracker (magenta) to visualize mitochondrial morphology. Scale bars, 5 µm. (L) Comparison of mitochondrial structure by confocal versus STED super-resolution microscopy in RAW 264.7 cells treated with DMSO or BT and stained with PK Mito. Scale bars, 2 µm. (M) Transmission electron micrographs of mitochondria in RAW 264.7 cells treated with DMSO or BT. (N) In vitro cGAS enzymatic assays: LC-MS quantification of cGAMP production by recombinant cGAS incubated with dsDNA in the presence of BT (25 or 100 µM). (O) LC-MS analysis of cGAMP production by cGAS incubated with BT (25 or 100 µM) in the absence of exogenous DNA. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Recombinant human cGAS protein were purchased from MedChemExpress (catalog no. HY-P72337).

Techniques: Binding Assay, Recombinant, Quantitative RT-PCR, Knock-Out, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Infection, Western Blot, In Vivo, Staining, Comparison, Super-Resolution Microscopy, Transmission Assay, In Vitro, Liquid Chromatography with Mass Spectroscopy, Incubation