murine macrophage cell line raw264 7  (ATCC)

Journal: Bioactive Materials

Article Title: A foam cell-targeted lipophagy restoration strategy stabilizes vulnerable atherosclerotic plaques

doi: 10.1016/j.bioactmat.2026.02.041

Figure Lengend Snippet: In vivo photoacoustic imaging and analysis of the vulnerability of atherosclerotic plaque. ( A - G ) Ex vivo distribution of HMCN@Cy5.5 , Scr-HMCN@Cy5.5 , and OPN-HMCN@Cy5.5 in various organs—specifically the aorta ( B ), heart ( C ), liver ( D ), spleen ( E ), lung ( F ), and kidney ( G )—from apoE −/− mice at 0, 6, 12, and 24 h post-intravenous injection (n = 3). ( H ) Confocal images demonstrate the colocalization of OPN with CY5.5-labeled nanoparticles in aortic roots (n = 6, scale bars, 200 μm). ( I ) Quantitative analysis of the relative MFI of OPN and CY5.5 in different treatment groups. ( J , K ) Photoacoustic images and quantitative analysis of signal intensities of atherosclerotic plaque in carotid arteries of both healthy and atherosclerosis mice (n = 3). For each animal, longitudinal PA imaging was performed on the same carotid artery at predefined anatomical landmarks across different time points. Photoacoustic images were acquired with depth calibration based on acoustic time-of-flight measurements, converting ultrasound echo delay into depth using the predefined sound velocity in soft tissue. A calibrated depth scale bar is shown in each image, with an effective imaging depth of approximately 7 mm. ( L , M ) Pathological staining of atherosclerotic plaques in the carotid artery and aortic arch includes ORO and Masson staining (scale bar = 200 μm), as well as α -SMA, and CD68 fluorescent staining (scale bar = 100 μm each). ( N - Q ) The statistical analysis of ( N ) ORO staining (namely the percentage of LD area), ( O ) Masson staining (namely the percentage of collagen fiber area), ( P ) α -SMA fluorescent staining (namely the percentage of smooth muscle cell area) and ( Q ) CD68 fluorescent staining (namely the percentage of macrophage-derived foam cell area). ( R ) Vulnerability scores of aortic arch and carotid artery plaques. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗∗ P < 0.0001.

Article Snippet: Mouse macrophage cell line (RAW264.7) was obtained from the American Type Culture Collection, USA.

Techniques: In Vivo, Imaging, Ex Vivo, Injection, Labeling, Staining, Derivative Assay

Journal: Bioactive Materials

Article Title: A foam cell-targeted lipophagy restoration strategy stabilizes vulnerable atherosclerotic plaques

doi: 10.1016/j.bioactmat.2026.02.041

Figure Lengend Snippet: In vivo atherosclerosis reversal. ( A ) Schematic illustration of the experimental timeline and treatment strategy for establishing a mature, vulnerable atherosclerosis model and evaluating therapeutic interventions. Mice were fed a high-fat diet (HFD) for 12 weeks and then divided into five groups (HFD+ 12W, Saline HFD+, OPN-HMCN@MLT HFD+, Saline HFD−, and OPN-HMCN@MLT HFD−). Except for the HFD+ 12W group, the remaining groups were further maintained for an additional 4 weeks under either HFD or non-HFD conditions with the indicated treatments. ( B , C ) Images of en face ORO-stained aortas ( B ) and quantitative analysis of ORO-positive regions ( C ) from mice subjected to different treatments and diets (n = 6, scale bar: 5 mm). ( D ) Aortic root sections stained by ORO, H&E, α-SMA antibody, Masson's trichrome, CD68 antibody, and MMP-9 antibody, respectively, following various therapeutic procedures (n = 6, scale bar: 500 μm). ( E - J ) Quantitative data of lipid accumulation ( E ), necrotic core area ( F ), collagen area ( G ), MMP-9 level ( H ), VSMC area ( I ), and macrophage-foam cell area ( J ) in aortic root sections. ( K ) Vulnerability scores of aortic root plaque. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: Mouse macrophage cell line (RAW264.7) was obtained from the American Type Culture Collection, USA.

Techniques: In Vivo, Saline, Staining

Journal: Bioactive Materials

Article Title: A foam cell-targeted lipophagy restoration strategy stabilizes vulnerable atherosclerotic plaques

doi: 10.1016/j.bioactmat.2026.02.041

Figure Lengend Snippet: In vivo anti-atherosclerosis effects. ( A ) Diagram illustrating the treatment protocol for apoE −/− mice. ( B , C ) En face ORO staining images and quantitative analysis of the lesion area of aortic lesion areas in apoE −/− mice following various treatments (n = 6, scale bar: 5 mm). ( D ) Quantification of the reduction ratio (versus model) of ORO-positive area to the entire aorta. ( E ) Cross-sectional images of ORO-stained aortic root (scale bars, 500 μm) and brachiocephalic artery (scale bars, 200 μm). n = 6. ( F and G ) Quantitative analysis of the aortic root lesion area ( F ) and the reduction ratio (versus model) of ORO-positive area to the aortic root ( G ). ( H ) Aortic root sections stained by H&E, α-SMA antibody, Masson's trichrome, CD68 antibody, MMP-9 antibody, and OPN antibody, respectively, following various therapeutic procedures (n = 6, scale bar: 500 μm). ( I-M ) Quantitative data of necrotic core area ( I ), collagen area ( J ), VSMC area ( K ), macrophage-foam cell area ( L ), and MMP-9 level ( M ) in aortic root sections. ( N ) Representative TEM images of LDs in the aortic root and arch of apoE −/− mice following various treatments (scale bar: 1 μm). The green arrow indicates elastic fibers. ( O-R ) Quantification of lipid droplet number and average area per cell section, n = 6. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: Mouse macrophage cell line (RAW264.7) was obtained from the American Type Culture Collection, USA.

Techniques: In Vivo, Staining

Journal: Bioactive Materials

Article Title: A foam cell-targeted lipophagy restoration strategy stabilizes vulnerable atherosclerotic plaques

doi: 10.1016/j.bioactmat.2026.02.041

Figure Lengend Snippet: Schematic of the anti-atherosclerotic mechanism of OPN-HMCN@MLT. ( A ) The study commenced with the synthesis of mesoporous carbon nanospheres (MCN) functionalized with an OPN-binding peptide and hyaluronic acid to construct the OPN-HMCN nanoplatform. The OPN-binding peptide was designed to recognize OPN enriched in the extracellular matrix and on the surface of foam cells, thereby enabling selective accumulation in OPN-rich pathological regions. Following OPN recognition, OPN-HMCN@MLT undergoes CD44-dependent endocytosis. Melatonin (MLT), a lipid autophagy–promoting agent, was subsequently encapsulated within the nanocarrier to form OPN-HMCN@MLT. Firstly, the released MLT can bind to and upregulate the expression of PPARα and PPARγ, which then promote the expression of downstream genes (ABCA1, ABCG1, ACOX-1, and CTP1A) and trigger the lipophagy. ( B ) Subsequently, its lipophagy-enhancing effects, including ABCA1/G1-mediated cholesterol efflux and CTP1A/ACOX-1-mediated mitochondrial fatty acid oxidation, were studied to confirm the reversal of foam cell formation. ( C ) These effects eventually promote foam cells to reverse into macrophages. Abbreviations: MCN, mesoporous carbon nanoparticle; OPN, osteopontin; MLT, melatonin; LDL, low-density lipoprotein; ox-LDL, oxidized low-density lipoprotein; PA, Photoacoustic.

Article Snippet: Mouse macrophage cell line (RAW264.7) was obtained from the American Type Culture Collection, USA.

Techniques: Binding Assay, Construct, Expressing

Journal: STAR Protocols

Article Title: Protocol for pro-inflammatory microRNA motif discovery using machine learning

doi: 10.1016/j.xpro.2026.104467

Figure Lengend Snippet: Microscopic images of RAW 264.7 cells in 96-well plate before starvation and transfection (related to step 10) (A) 70% confluency. (B) <50% confluency. Scale bars represent 100 μm.

Article Snippet: RAW 264.7 mouse macrophage cell line , ATCC , Cat#TIB-71; RRID: CVCL_0493.

Techniques: Transfection

Journal: iScience

Article Title: Fabrication of RADA32/Ngf_EE/MSCs composite hydrogel and its protective mechanism against radiation-induced ototoxicity

doi: 10.1016/j.isci.2026.115723

Figure Lengend Snippet: Immunomodulatory mechanism of RNM composite gel (A and B) Flow cytometric analysis of CD86 and CD206 expression in RAW264.7 macrophages after irradiation and co-culture with RN, MSCs, or RNM composite gel in a transwell system (macrophages in lower chamber). Data are represented as the mean ± SEM ( N = 3, t test). (C) Immunofluorescence staining of F4/80 (red) on cochlear sections. Scale bars, 50 μm (a, spiral ganglion; b, basilar membrane; c, stria vascularis; d, spiral ligament). (D) Apoptosis of HEI-OC1 cells analyzed by flow cytometry after radiation exposure and intervention. Data are represented as the mean ± SEM ( N = 3, t test). (E) Expression level of p-p65, a key marker of NF-κB pathway activation, in macrophages after radiation exposure and drug intervention. Data are represented as the mean ± SEM ( N = 3, t test). Significant differences between the groups are indicated by ∗ for p < 0.05, ∗∗ for p < 0.01, ∗∗∗ for p < 0.001, and ∗∗∗∗ for p < 0.0001.

Article Snippet: Mouse bone marrow-derived mesenchymal stem cells (MSCs) and the mouse monocyte/macrophage cell line RAW264.7 were purchased from Procell Life Science & Technology Co., Ltd.

Techniques: Expressing, Irradiation, Co-Culture Assay, Immunofluorescence, Staining, Membrane, Flow Cytometry, Marker, Activation Assay

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Analysis of macrophage activation in the uterine tissue of cows with endometritis. (A, B) Representative immunofluorescence (IF) staining images (A) and quantitative analysis (B) of iNOS (M1 marker, red) in endometrial tissues from healthy cows and cows with endometritis. Nuclei were counterstained with DAPI (blue). (C, D) Representative IF staining images (C) and quantitative analysis (D) of Arg1 (M2 marker, red) in endometrial tissues. (E, F) Relative mRNA expression levels of iNOS (E) and Arg1 (F) in endometrial tissues, as determined by qPCR. (G, H) Relative expression levels of IL-1β, IL-6 and TNF-α in endometrial tissues, as determined by IHC. (I, J) Relative mRNA expression levels of IL-1β (I) and IL-6 (J) in endometrial tissues, as determined by qPCR. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The macrophage cell line (BoMac) is a transformed macrophage cell line originally described by Stabel and Stabel [ ], and the cells were maintained in DMEM supplemented with 10% fetal bovine serum.

Techniques: Activation Assay, Immunofluorescence, Staining, Marker, Expressing

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Exosomes from LPS-stimulated EECs induce pro-inflammatory macrophage activation. (A) Schematic diagram of the experimental setup for exosome uptake. (B) Fluorescence microscopy images showing the uptake of PKH67-labeled exosomes (green) by macrophages. Cytoskeleton was stained with Phalloidin (red), and nuclei were stained with DAPI (blue). (C) Western blotting analysis of phosphorylated NF-κB p65 (p-p65) in macrophages treated with Control-exo or LPS-exo. (D, E) Representative immunofluorescence (IF) staining images (D) and quantitative analysis (E) of iNOS (greed) in macrophages. (F, G) Representative IF staining images (F) and quantitative analysis (G) of Arg1 (red) in macrophages. (H) Schematic diagram of co-culture experiments. (I, J) Relative mRNA expression levels of iNOS (I) and Arg1 (J) in macrophages after co-culture with EECs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The macrophage cell line (BoMac) is a transformed macrophage cell line originally described by Stabel and Stabel [ ], and the cells were maintained in DMEM supplemented with 10% fetal bovine serum.

Techniques: Activation Assay, Fluorescence, Microscopy, Labeling, Staining, Western Blot, Control, Immunofluorescence, Co-Culture Assay, Expressing

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Transcriptomic profiling reveals significant enrichment of lncRNA OTUD6B-AS1 in exosomes derived from LPS-stimulated EECs. (A, B) LPS-exo was treated with RNase A alone or in combination with Triton X-100 for 4 h, and then co-incubated with macrophages. Relative expression levels of iNOS (A) and Arg1 (B) in macrophages. (C) Schematic overview of the RNA sequencing and analysis workflow. (D) Volcano plot showing differentially expressed lncRNAs in LPS-exo compared to Control-exo. (E, F) Gene Ontology (GO) biological process enrichment analysis (E) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (F) of the differentially expressed lncRNAs. (G) qPCR validation of the 6 upregulated lncRNAs in Control-exo and LPS-exo. (H) LPS-exo was treated with RNase A alone or in combination with Triton X-100 for 4 h, and then co-incubated with macrophages. Relative mRNA expression level of lncRNA OTUD6B-AS1 in macrophages. (I) A proposed competing endogenous RNA (ceRNA) network involving lncRNA OTUD6B-AS1, miR-128, and Notch2. (J) Relative mRNA expression level of lncRNA OTUD6B-AS1 in control and LPS-stimulated EECs. (K, L) RNA fluorescence in situ hybridization (RNA-FISH) showing the subcellular localization of lncRNA OTUD6B-AS1 (red) in EECs (K) and its quantitative cytoplasmic/nuclear distribution (L). Nuclei were stained with DAPI (blue). (M – P) Relative mRNA expression levels of lncRNA OTUD6B-AS1 (M − O) and miR-128 (P) in endometrial tissues from healthy cows and cows with endometritis, as determined by qPCR (O, P) and RNA-FISH (M) with quantification (N). (Q) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 protein levels in endometrial tissues. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The macrophage cell line (BoMac) is a transformed macrophage cell line originally described by Stabel and Stabel [ ], and the cells were maintained in DMEM supplemented with 10% fetal bovine serum.

Techniques: Derivative Assay, Incubation, Expressing, RNA Sequencing, Control, Biomarker Discovery, Fluorescence, In Situ Hybridization, Staining, Western Blot

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: EECs-derived exosomes induce pro-inflammatory macrophage activation via delivery of lncRNA OTUD6B-AS1. (A) Relative mRNA expression level of lncRNA OTUD6B-AS1 in macrophages treated with Control-exo or LPS-exo. (B, C) RNA-FISH images (B) and quantitative analysis (C) showing lncRNA OTUD6B-AS1 (red) transfer to macrophages after co-culture with Control-exo or LPS-exo. Nuclei were stained with DAPI (blue). (D) Relative mRNA expression level of lncRNA OTUD6B-AS1 in macrophages after transfection with lncRNA OTUD6B-AS1 overexpression plasmids (OE-lncRNA) or control plasmids (OE-NC). ( E – I) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (E), along with immunofluorescence (IF) quantitative analysis of iNOS (F, G) and Arg1 (H, I) protein levels in macrophages after transfection with OE-lncRNA or OE-NC. (J – N) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (J), along with IF quantitative analysis of iNOS (K, L) and Arg1 (M, N) protein levels in macrophages after lncRNA OTUD6B-AS1 knockdown (si-lncRNA) or control treatment (si-NC). (O) Relative mRNA expression level of lncRNA OTUD6B-AS1 in exosomes isolated from lncRNA OTUD6B-AS1-knockdown LPS-stimulated EECs (si-lncRNA-LPS-exo) or exosomes from siRNA NC-transfected LPS-stimulated EECs (si-NC-LPS-exo). (P – S) IF quantitative analysis of iNOS (P, Q) and Arg1 (R, S) protein levels in macrophages treated with si-lncRNA-LPS-exo or si-NC-LPS-exo. (T) Relative mRNA expression levels of iNOS and Arg1 in macrophages treated with exosomes isolated from control EECs overexpressing lncRNA OTUD6B-AS1 (OE-lncRNA-Control-exo) or exosomes from control plasmids-transfected EECs (OE-NC-Control-exo). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The macrophage cell line (BoMac) is a transformed macrophage cell line originally described by Stabel and Stabel [ ], and the cells were maintained in DMEM supplemented with 10% fetal bovine serum.

Techniques: Derivative Assay, Activation Assay, Expressing, Control, Co-Culture Assay, Staining, Transfection, Over Expression, Western Blot, Immunofluorescence, Knockdown, Isolation

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: lncRNA OTUD6B-AS1 acts as a ceRNA by sponging miR-128 to facilitate pro-inflammatory macrophage activation. (A) Relative mRNA expression level of miR-128 in macrophages treated with Control-exo or LPS-exo. (B) Luciferase reporter assay in HEK293T cells co-transfected with wild-type (WT) or mutant (MUT) lncRNA OTUD6B-AS1 reporter plasmids and miR-128 mimic or mimic NC. (C) RNA pull-down detection of the enrichment of miR-128 to lncRNA OTUD6B-AS1. (D) Ago2 RIP assay analysis of the enrichment of lncRNA OTUD6B-AS1 pulled-down from the Ago2 protein. (E) Relative mRNA expression level of miR-128 in macrophages transfected with OE-NC or OE-lncRNA. (F – J) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (F), along with immunofluorescence (IF) quantitative analysis of iNOS (G, H) and Arg1 (I, J) protein levels in macrophages co-transfected with OE-lncRNA and miR-128 mimic or mimic NC. (K, L) Relative mRNA expression levels of IL-1β (K) and IL-6 (L) in macrophages co-transfected with OE-lncRNA and miR-128 mimic or mimic NC. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The macrophage cell line (BoMac) is a transformed macrophage cell line originally described by Stabel and Stabel [ ], and the cells were maintained in DMEM supplemented with 10% fetal bovine serum.

Techniques: Activation Assay, Expressing, Control, Luciferase, Reporter Assay, Transfection, Mutagenesis, Western Blot, Immunofluorescence

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: Notch2 mediates the regulatory effect of the lncRNA OTUD6B-AS1/miR-128 axis on macrophage activation. (A) Predictive analysis of miR-128 targets using multiple databases. (B) Western blotting analysis of Notch2 protein levels in macrophages treated with Control-exo or LPS-exo. (C) Western blotting analysis of Notch2 protein levels in macrophages transfected with OE-NC or OE-lncRNA. (D – H) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (D), along with immunofluorescence (IF) quantitative analysis of iNOS (E, F) and Arg1 (G, H) protein levels in macrophages treated with OE-NC or OE-lncRNA and the Notch2 inhibitor DAPT. (I) Luciferase reporter assay in HEK293T cells co-transfected with WT or MUT Notch2 3′UTR reporter plasmids and miR-128 mimic or mimic NC. (J, K) Relative protein (J) and mRNA (K) expression levels of Notch2 in macrophages transfected with miR-128 mimic or mimic NC. (L – P) Western blotting analysis of Notch2, RBP-Jκ, and p-p65 (L), along with IF quantitative analysis of iNOS (M, N) and Arg1 (O, P) protein levels in macrophages co-treated with miR-128 inhibitor or inhibitor NC and DAPT. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: The macrophage cell line (BoMac) is a transformed macrophage cell line originally described by Stabel and Stabel [ ], and the cells were maintained in DMEM supplemented with 10% fetal bovine serum.

Techniques: Activation Assay, Western Blot, Control, Transfection, Immunofluorescence, Luciferase, Reporter Assay, Expressing

Journal: Materials Today Bio

Article Title: Exosomal lncRNA OTUD6B-AS1 as a pathogenic nanocarrier promotes inflammatory macrophage polarization in endometritis via a targetable ceRNA circuit

doi: 10.1016/j.mtbio.2026.103027

Figure Lengend Snippet: A proposed model illustrating the exosome-mediated lncRNA OTUD6B-AS1/miR-128/Notch2 axis in aggravating endometritis. Upon LPS-induced damage, endometrial epithelial cells (EECs) release increased exosomes carrying elevated levels of lncRNA OTUD6B-AS1. These exosomes are taken up by endometrial macrophages. The transferred lncRNA OTUD6B-AS1 acts as a molecular sponge to sequester miR-128, leading to the derepression and upregulation of its target gene, Notch2. The enhanced Notch2 signaling subsequently promotes macrophage polarization towards a pro-inflammatory M1 phenotype, characterized by increased NF-κB activation and iNOS expression, thereby exacerbating endometrial inflammation and tissue damage.

Article Snippet: The macrophage cell line (BoMac) is a transformed macrophage cell line originally described by Stabel and Stabel [ ], and the cells were maintained in DMEM supplemented with 10% fetal bovine serum.

Techniques: Activation Assay, Expressing

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: The murine macrophage cell line RAW 264.7 (ATCC TIB-71, USA) was cultured in high-glucose DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C and 5% CO2.

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

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: The murine macrophage cell line RAW 264.7 (ATCC TIB-71, USA) was cultured in high-glucose DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C and 5% CO2.

Techniques: Activation Assay, Derivative Assay, Western Blot, Control, Gene Expression, Expressing, Inhibition, Cell Culture, Recombinant

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 2′3′-cGAMP stimulation (5 µg/mL) on macrophage- and interferon-related gene expression in BMDMs after 24 h. Data are normalized to the unstimulated control. (B) Cytokine release (IL-6, TNF-α, IL-10 and IFN-β) by 2′3′-cGAMP-stimulated BMDMs after 24 h. (C) Expression of surface activation markers (TLR2, MHC class II and CD80) in BMDMs 24 h after stimulation with 2′3′-cGAMP. Data are presented as ratios of +cGAMP to –cGAMP. (D) MHC class II surface expression in RAW 264.7 cells 24 h after stimulation with 50 ng/mL RANKL, 10 µg/mL 2′3′-cGAMP or the combination of both. Data are normalized to the unstimulated control. (E) RANK surface expression in RAW 264.7 cells 24 h after stimulation with 50 ng/mL RANKL, 10 µg/mL 2′3′-cGAMP or the combination of both. Left: RANK levels normalized to the unstimulated control. Right: RANK expression presented as ratios of +cGAMP to –cGAMP. Different symbols and dotted lines indicating independent experiments, respectively. (A-E) BMDMs were cultured in the presence of 25 ng/mL recombinant mouse M-CSF throughout all experiments. Bar graphs show mean ± SEM with individual data points. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc test (n = 3). Mϕ: macrophage.

Article Snippet: The murine macrophage cell line RAW 264.7 (ATCC TIB-71, USA) was cultured in high-glucose DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C and 5% CO2.

Techniques: Gene Expression, Control, Expressing, Activation Assay, Cell Culture, Recombinant