Journal: bioRxiv

Article Title: cGAS–STING induced IFN-β acts as a dual regulator of osteoclastogenesis via direct and osteoblast-mediated mechanisms

doi: 10.64898/2026.05.09.724040

Figure Lengend Snippet: (A) Effect of cGAS activation by G3-YSD complexed in LyoVec™ (YSD/LV; BMDMs: 250 ng/mL, RAW 264.7: 500 ng/mL) on RANKL-mediated osteoclast formation. Representative images of osteoclasts derived from BMDMs (left) and quantification of relative osteoclast numbers per well in BMDMs and RAW 264.7 cells (right). (B+C) Gene expression analysis of interferon-related genes (B) and osteoclast-associated genes (C) 48 h after stimulation with G3-YSD complexed in LyoVec™ (YSD/LV; BMDMs: 250 ng/mL, RAW 264.7: 500 ng/mL) in the presence or absence of 50 ng/mL RANKL. Data are normalized to the unstimulated control. (D–G) Effect of cGAS inhibition using RU.521 (10 µg/mL in DMSO) on osteoclast formation in RAW 264.7 cells. (D) Quantification of relative osteoclast numbers per well. (E) Gene expression analysis of interferon-related and osteoclast-associated genes 48 h after cGAS inhibition in the presence of 50 ng/mL RANKL. Data are normalized to the unstimulated control. (F) Time-dependent effects of cGAS inhibition, with inhibitor (RU.521, 10 µg/mL in DMSO) added throughout differentiation (“both”), during early stages (first 3 days) or during late stages (days 3–5/6). (G) Pre-inhibition of cGAS by treatment with RU.521 (10 µg/mL in DMSO) 24 h prior to RANKL stimulation. The inhibitor was removed before 50 ng/mL RANKL was added. Left: relative osteoclast numbers per well. Right: gene expression analysis of interferon- and macrophage-related genes and osteoclast-associated genes after 24 h cGAS inhibition followed by 48 h RANKL treatment. Data are normalized to the DMSO pre-treated RANKL control. (A-G) BMDMs were cultured in the presence of 25 ng/mL recombinant mouse M-CSF throughout all experiments. Osteoclast numbers per well are shown relatively to the RANKL control. Heatmaps display mean values, and bar graphs show mean ± SEM with individual data points. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc test (n = 3). RL: RANKL; LV: LyoVec™ transfection agent.

Article Snippet: Where indicated, cells were treated with: cGAS agonist G3-YSD (RAW 264.7: 500 ng/mL; BMDMs: 250 ng/mL) complexed with LyoVecTM (1:100, 15 min pre-incubation), cGAS inhibitor RU.521 (10 μg/mL in DMSO) added 3 h prior to stimulation; STING agonists 2′3′-cGAMP (RAW: 10 μg/mL; BMDMs: 5 μg/mL) or diABZI (0.01–10 μg/mL), STING inhibitor H-151 (40 or 400 ng/mL in DMSO) added 2 h prior to stimulation (all InvivoGen, USA).

Techniques: Activation Assay, Derivative Assay, Gene Expression, Control, Inhibition, Cell Culture, Recombinant, Transfection