Journal: Bioactive Materials

Article Title: Bioengineered titanium implants functionalized with aptamer-valproic acid conjugates orchestrate macrophage programming and mesenchymal stem cell homing for improved osseointegration

doi: 10.1016/j.bioactmat.2026.05.055

Figure Lengend Snippet: A) Live/Dead staining of RAW 264.7 macrophages cultured on different material surfaces for 72 h, where dead cells were stained red and live cells were stained green. B) Cytoskeleton staining morphology of RAW 264.7 grown on different material surfaces for 72 h. C, D, F) Fluorescence images and flow cytometry analysis of intracellular ROS in RAW 264.7 cells with DCFH-DA probe. E) Proliferation of RAW 264.7 macrophages on different material surfaces assessed by CCK-8 assay. n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Created in BioRender.

Article Snippet: The murine macrophage cell line RAW 264.7 (Procell Life Science, China) was cultured in high-glucose DMEM (Gibco, USA) containing 10% FBS and employed for immunomodulation studies.

Techniques: Staining, Cell Culture, Fluorescence, Flow Cytometry, CCK-8 Assay

Journal: Bioactive Materials

Article Title: Bioengineered titanium implants functionalized with aptamer-valproic acid conjugates orchestrate macrophage programming and mesenchymal stem cell homing for improved osseointegration

doi: 10.1016/j.bioactmat.2026.05.055

Figure Lengend Snippet: A, F) IF staining of iNOS and CD206 in RAW 264.7 macrophages. B-E) Secretion of inflammation-related proteins in RAW 264.7 macrophages. G-J) Relative mRNA expression of inflammation-related genes in RAW 264.7 macrophages. n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The murine macrophage cell line RAW 264.7 (Procell Life Science, China) was cultured in high-glucose DMEM (Gibco, USA) containing 10% FBS and employed for immunomodulation studies.

Techniques: Staining, Expressing

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: 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