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 STING activation by 2′3′-cGAMP (BMDMs: 5 µg/mL, RAW 264.7: 10 µg/mL) on RANKL-mediated osteoclast formation. 2’3’-cGAMP were given throughout the differentiation or for BMDMs also during late stages (days 3–5/6). 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) Immunoblot analysis of NFATc1 protein levels of RAW 264.7 cells following RANKL stimulation (50 ng/mL) in the presence or absence of 10 µg/mL 2′3′-cGAMP. GAPDH served as a loading control. (C) Gene expression analysis of interferon-related, macrophage-related and osteoclast-associated genes of RAW 264.7 cells 48 h after stimulation with 50 ng/mL RANKL with or without 10 µg/mL 2′3′-cGAMP. Data are presented as ratios of +cGAMP to –cGAMP. (D) Effect of STING activation by diABZI (0.01 until 10 µg/mL) on 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). (E) Induction of the interferon-responsive gene Isg15 following STING activation with diABZI (0.01 until 10 µg/mL) in BMDMs (upper) and RAW 264.7 cells (lower). Data are normalized to the unstimulated control. (F) Effect of STING activation by diABZI (0.01 until 10 µg/mL) on RANKL-induced NFATc1 expression at mRNA and protein levels after 24 h in RAW 264.7 cells. (G) Gene expression analysis of osteoclast-associated genes 48 h after stimulation with diABZI (0.01 until 10 µg/mL) and 50 ng/mL RANKL in RAW 264.7 cells. Data are normalized to the unstimulated control. (H) Effect of STING inhibition using H-151 (RAW 264.7: 40 or 400 ng/mL in DMSO, BMDMs: 400 ng/mL in DMSO) on osteoclast formation. Left and middle: quantification of relative osteoclast numbers per well upon continuous inhibitor treatment. Right: time-dependent effects of STING inhibition with inhibitor added during early stages (first 3 days) or late stages (days 3–5/6) of differentiation. 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.

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, Western Blot, Control, Gene Expression, Expressing, Inhibition, Cell Culture, Recombinant

Journal: bioRxiv

Article Title: It’s Not Rewarding for Mitochondria: Dopamine-Induced Mitochondrial Dysfunction Activates cGAS-STING to Drive IL-6 Secretion in Macrophages

doi: 10.64898/2026.04.23.719926

Figure Lengend Snippet: (A) Cells were pretreated (t = −1 hr) with BAY 11-7082 (10 μM) to inhibit NFκB activation. IL-6 secretion was measured 24 hrs after dopamine treatment using AlphaLISA, demonstrating that dopamine-induced IL-6 secretion is mediated through the NFκB pathway, (Biological replicates: N=19). Data are presented as box and whisker plot (min to max). See and . (B-F) Cells were treated with dopamine in a time-course experiment (5, 15, 30, 60, and 360 minutes) and assessed for cGAS protein expression (normalized to total protein expression, Biological replicate: N=5-14) as well as phosphorylation of STING (Biological replicate: N=7-9), TBK1 (Biological replicate: N=5-7), and IRF3 (Biological replicate: N=5), (normalized to the total expression level of each protein) by western blotting. Individual values are shown with line demonstrating mean. See and . (G-I) Cells were pretreated (t = −1 hr) with G-140 (1 μM) or H-151 (1 μM) to inhibit the cGAS-STING pathway, and the inhibitory effects were validated by western blotting. Cells were then treated with dopamine (t = 0). IL-6 secretion was measured 24 hrs post-treatment using AlphaLISA (Biological replicate: G-140:N=14 and H-151: N=10), demonstrating that activation of the cGAS-STING pathway contributes to dopamine-induced IL-6 secretion. Data are presented as box and whisker plot (min to max). Paired comparisons were analyzed using a paired t -test (non-parametric: Wilcoxon test) in panel C-F or paired one-way ANOVA (non-parametric: Friedman test) in panel A, H and I. Significance levels are indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Article Snippet: Dopamine hydrochloride (DA; #H8502, Sigma-Aldrich), lipopolysaccharide from Escherichia coli O55:B5 (LPS; #L2880, MilliporeSigma), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; #0453, Tocris), 2′3′-cGAMP (#HY-12512, MedChemExpress), Mdivi-1 (#HY-15886, MedChemExpress), G140 (#HY-133916, MedChemExpress), H-151 (#HY-112693, MedChemExpress), ethidium bromide (EtBr; #HY-D0021, MedChemExpress), BAY 11-7082 (#S2913, Selleck Chemicals) were purchased from the indicated vendors.

Techniques: Activation Assay, Whisker Assay, Expressing, Phospho-proteomics, Western Blot