Journal: Bioactive Materials

Article Title: Chronic inflammation-responsive hydrogel restores myeloid-T cell crosstalk to reinvigorate antitumor immunity against metastatic colorectal cancer

doi: 10.1016/j.bioactmat.2026.03.012

Figure Lengend Snippet: PGE2 blockade modulates immune cell phenotypes in antitumor resp onses. (A) Inflammatory gene expression across cancer types (GEPIA2 database). (B) Gene expression of Il1b , Cxcl8 , and Lif in colon adenocarcinoma (COAD) tumor tissue and normal tissue (GEPIA2 database). (C and D) Correlation between Ptgs2 and inflammatory genes in various cancers (C) and COAD (D) (TIMER 2.0). (E) Schematic of immune cells co-incubated with CXB treated tumor conditional medium (TCM) (Source material from BioRender). (F and G) Cell viability (F) and Cell cycle arrest (G) detection of CT26 tumor cells treated with gradient concentrations of CXB; n = 3. (H) PGE2 concentration in CT26 cell supernatants; n = 3. (I) The proportion of CD103 + DC within BMDCs after CXB treatments in vitro ; n = 3. (J and K) Maturation (J) and Antigen processing capability (K) on BMDCs; n = 3. (L – N) Flow charts of CD86 or CD206 expression on Raw 264.7 cells (L). Quantification of CD86 (M) and CD206 (N) expression on Raw 264.7 cells; n = 3. (O and P) Flow charts (O) and Quantification (P) of CD69 and CD137 expression on splenic T cells exposed to CXB-pretreated TCM; n = 3. (Q) IFN-γ secretion by T cells co-cultured with CXB-pretreated TCM; n = 3. Data are presented as mean ± SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Significance was calculated using One-way ANOVA.

Article Snippet: CT26 cells and Raw 264.7 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Gene Expression, Incubation, Concentration Assay, In Vitro, Expressing, Cell Culture

Journal: Bioactive Materials

Article Title: Chronic inflammation-responsive hydrogel restores myeloid-T cell crosstalk to reinvigorate antitumor immunity against metastatic colorectal cancer

doi: 10.1016/j.bioactmat.2026.03.012

Figure Lengend Snippet: TRANS modulates macrophage polarization and promotes antigen present ation. (A) Uniform manifold approximation and projection (UMAP) visualization of myeloid cells in the TME following PBS or TRANS treatment. (B) UMAP clusters of myeloid cells. (C) Cell-type-specific marker genes expression of different clusters. (D) Proportion distribution of myeloid cell clusters. (E) KEGG enrichment in M1 macrophages (TRANS vs PBS). (F) GSEA of NOD-like receptor and JAK-STAT signaling pathways in M1. (G) KEGG enrichment in M2 macrophages (TRANS vs PBS). (H) Glycolysis/Gluconeogenesis and antigen processing/presentation in M2. (I) Scheme of M0 macrophages treated with different TCM. (J) M1 and M2 polarization from M0 macrophages under various treatments; n = 3. (K) STAT1 expression in Raw 264.7 cells. (L) Scheme of M2 macrophages treated with different TCM. (M) Repolarization of M2 macrophages under different treatments; n = 3. (N) H-2K b /SIINFEKL + macrophages in vitro ; n = 3. (O and P) Relative expression of antitumor genes (O) and inflammatory genes (P) in macrophages under different treatments in vitro ; n = 3. (Q) CD69 expression on T cells after co-incubating TCM-treated macrophages and anti-CD3/CD28-stimulated splenic T cells; n = 3. Data are presented as mean ± SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Significance of multiple-group comparisons was calculated using One-way ANOVA. Significance of two-group comparison was calculated using Student's t -test.

Article Snippet: CT26 cells and Raw 264.7 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Marker, Expressing, Protein-Protein interactions, In Vitro, Comparison

Journal: Bioactive Materials

Article Title: Near infrared enhanced palladium loaded siraitia grosvenorii carbon dots amplify mitophagy for acute lung injury immunotherapy

doi: 10.1016/j.bioactmat.2026.02.040

Figure Lengend Snippet: Schematic illustration of the preparation of CPs@SS31, and the corresponding in vivo therapy of acute lung injury via NIR enhanced ROS scavenging, inflammation inhibition, macrophage M2 polarization, and T cells immunoactivation, as well as specifically targeting mitochondria, activating mitochondrial function, and inducing mitophagy to reprogram lung redox homeostasis, and promote tissue repair.

Article Snippet: Cell viability testing : Mouse monocyte macrophages (RAW264.7, ATCC, USA) were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin (Solarbio, China).

Techniques: In Vivo, Inhibition

Journal: Bioactive Materials

Article Title: Smart microenvironment-adaptive nanocatalytic hydrogel for sequential antibacterial, anti-inflammatory, and regenerative therapy of biofilm-infected wounds

doi: 10.1016/j.bioactmat.2026.02.043

Figure Lengend Snippet: Characterization, and Cytocompatibility Validation of HCOC. (A) Schematic illustration of the development of HCOC. (B) FTIR spectrum of OSA, CMCS and OC hydrogel. (C) Time-dependent evolution of gelation of OC and HCOC. (D) SEM images of HCOC and EDS mapping images of C, N, O and Cu for HCOC. (E) FTIR spectra of HC, OC and HCOC. (F) Dynamic frequency sweep measurements of OC and HCOC. (G) Frequency-dependent viscoelastic behavior of OC and HCOC. (H) Alternating strain sweep with alternating strains of 1% and 1000% at 100s intervals and (I) Self-healing behavior of HCOC. (J) Live/dead staining showing the metabolic activity of L929 and RAW 264.7 cells after treatment with HCOC for 48 h. Rates of proliferation of (K) L929 cells and (L) RAW 264.7 cells after treatment with PBS or HCOC. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

Article Snippet: L929 and RAW 264.7 cells were purchased from the American-Type Culture Collection (ATCC).

Techniques: Biomarker Discovery, Staining, Activity Assay

Journal: Bioactive Materials

Article Title: Smart microenvironment-adaptive nanocatalytic hydrogel for sequential antibacterial, anti-inflammatory, and regenerative therapy of biofilm-infected wounds

doi: 10.1016/j.bioactmat.2026.02.043

Figure Lengend Snippet: Macrophage immunoregulation, cell proliferation and angiogenesis in vitro triggered by HCOC. (A) Localization of CD86 (orange, M1 marker) and CD206 (green, M2 marker) in RAW 264.7 macrophages after stimulation (24 h) with different test materials (PBS, 40 ng/mL IL-4, 2 mg/mL Cu 5.4 O, 2 mg/mL HAs, 2 mg/mL HC, 2 mg/mL HCOC). (B) Flow cytometric analysis quantifying CD86 + and CD206 + cell populations after 24 h inflammatory stimulation and different concentrations of HCOC. (C) Western blot analysis of key signaling proteins and phenotypic markers in LPS-stimulated RAW264.7 macrophages treated with HCOC (0.5 or 1.0 mg/mL) or IL-4 (20 ng/mL). Protein levels were normalized to GAPDH or the corresponding total protein. (D–G) mRNA expression of (D) TNF-α, (E) Arg1, (F) Axin2, and (G) IL-10 measured by real-time RT-PCR. Data are expressed relative to the LPS-only control group and presented as mean ± SEM of three independent experiments. (H) Following H 2 O 2 pretreatment, L929 cell migration and (I) HUVEC tube formation ( in vitro angiogenesis) were assessed after treatment with varying HCOC concentrations over a 24-h period. Quantitative analysis of (J) L929 cell migration rate and the formed (K tube length and (L) branch points. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

Article Snippet: L929 and RAW 264.7 cells were purchased from the American-Type Culture Collection (ATCC).

Techniques: In Vitro, Marker, Western Blot, Expressing, Quantitative RT-PCR, Control, Migration

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 vitro evaluation of foam cell lipid accumulation and lipophagy activation following OPN-HMCN@MLT treatment. ( A - C ) ORO and BODIPY staining images and corresponding quantification of ORO and BODIPY positive areas of RAW264.7 cells under different stimulations (n = 5, scale bar for ORO: 100 μm, scale bar for BODIPY: 20 μm). ( D ) Bio-TEM images of RAW264.7 cells post various treatments (n = 5, scale bars 1.0 μm). Green arrows indicate nanoparticles. ( E , F ) Morphometric analysis determined the mean number and area (μm 2 ) of LDs per cell section. ( G ) Confocal images depicting lipophagy flux in foam cells following different treatments (n = 5 biological replicates, scale bars: 10 μm). ( H - J ) The quantities of acidified autophagosomes (GFP-RFP+), neutral autophagosomes (GFP + RFP+), and LDs labeled with BODIPY were measured per cell for each condition. (K to N) Representative Western blot images and quantitative analysis of LC3, LAMP1, and P62 expression in foam cells. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: RAW264.7 mouse macrophage cells (ATCC® TIB-71; RRID: CVCL_0493) and MCAECs (Procell, CP-M081) were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: In Vitro, Activation Assay, Staining, Labeling, Western Blot, 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 vitro examination of LD degradation in foam cells through fatty acid oxidation and cholesterol efflux. (A ) Schematic diagram of the LD degradation mechanism. ( B , C ) Confocal images and quantitative analysis of LDs colocalization with fatty acids in RAW264.7 cells following different treatments (n = 5, scale bars: 5 μm). ( D , E ) Confocal images of the colocalization of mitochondria with fatty acids and quantified data of fatty acids in RAW264.7 cells under different stimulations (n = 5, scale bars: 5 μm). ( F , G ) Confocal images illustrating mitochondrial colocalization with ATP and corresponding quantification of ATP levels in RAW264.7 cells post various treatments (n = 5, scale bars: 20 μm). ( H ) Diagram illustrating the incorporation of [U- 13 C] palmitic acid into the TCA cycle and the labeling pattern of derived metabolites (n = 3). ( I ) A PCA plot illustrates the cluster separation between the two groups (n = 3). ( J ) Heatmap showing differences in metabolites between the two groups (n = 3). ( K ) Normalized total labeling of each metabolite to [U- 13 C] palmitic acid (n = 3). ( L ) Proportion of (m + 2)-labeled TCA cycle metabolites derived from [U- 13 C] palmitic acid (n = 3). ( M - R ) The study quantified NBD-cholesterol accumulation ( M , P ) and cholesterol efflux facilitated by HDL ( N , O ) and apoA-I ( Q , R ) using confocal imaging across (n = 5, Scale bar = 50 μm). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: RAW264.7 mouse macrophage cells (ATCC® TIB-71; RRID: CVCL_0493) and MCAECs (Procell, CP-M081) were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: In Vitro, Labeling, Derivative Assay, Imaging

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: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: MTOC proteins and Golgi are recruited to the nuclear envelope in RAW264.7-derived osteoclasts. (A) Immunostaining of AKAP6 (green) and GM130 (magenta) together with DNA (DAPI) in RAW264.7-derived osteoclasts after 4 days of RANKL addition. Scale bars: 10 µm. Immunostaining of (B) PCNT (green), (C) CDK5RAP2 (green) and (D) PCM1 (green) and γ-tubulin (magenta) together with DNA (DAPI) in RAW264.7-derived osteoclasts after 4 days of RANKL addition. Arrows indicate centrosomes. Scale bars: 10 µm. Images representative of three experimental repeats.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Derivative Assay, Immunostaining

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: RAW264.7-derived osteoclasts exhibit a nuclear envelope MTOC alongside a functional centrosome. (A) Immunostaining of α-tubulin (red), AKAP6 (green), GM130 (magenta) and DNA (DAPI) in RAW264.7-derived osteoclasts differentiated for 4 days. (B) Immunostaining of EB-1 (green), γ-tubulin (magenta) and DNA (DAPI) in RAW264.7-derived osteoclasts treated with nocodazole (upper row) and after 1 min (middle row) or 2 min (bottom row) recovery at 37°C. In A and B, asterisks indicate the perinuclear microtubule cage and arrows denote centrosomal asters. (C) Immunostaining of α-tubulin (red), PCNT (green), GM130 (magenta), and DNA (DAPI) in 4 day RAW264.7-derived osteoclasts after 1.5 min of recovery from nocodazole-induced microtubule depolymerization. Asterisks denote the NE nucleation, arrows denote centrosomal nucleation and arrowheads denote Golgi nucleation. Scale bars: 10 µm. Images representative of three experimental repeats.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Derivative Assay, Functional Assay, Immunostaining

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: Nesprin-1α and AKAP6β isoforms predominate in RAW264.7-derived osteoclasts and form a functional LINC complex. (A) Immunostaining of AKAP6 (green), nesprin-1 (magenta) and DNA (DAPI) in RAW264.7 cells either non-differentiated (upper panels) or differentiated into osteoclasts for 4 days with 50 ng ml −1 of RANKL (lower panels). Non-differentiated cells lack detectable expression of both AKAP6 and nesprin-1 (also denoted with an arrow). Scale bars: 10 µm. (B) Heatmap of qPCR-derived RNA expression levels for Emr1 , Akap6 , Syne1a , Trap and Ctsk during osteoclast differentiation (days 1–4 of RANKL treatment) relative to non-differentiated precursor cells ( n =3). Expression values are normalized and represented as a percentage of maximal expression per gene using the viridis color map. (C) qPCR analysis of Syne1a (gray circles) and Syne1 giant (black triangles) mRNA expression during osteoclast differentiation (D1, D2, D3, D4, day 1–4). (D) qPCR analysis of Akap6a (gray circles) and Akap6b (black triangles) transcript levels at the indicated differentiation stages. Data in C and D are presented as mean±s.d. from three independent experiments, normalized to the non-differentiated condition. (E) Representative images of RAW264.7-derived osteoclasts treated with vehicle (water) or 5 μM DTT and co-stained with Sun2C (green), laminB1 (magenta) antibodies and DAPI. The Sun2C antibody recognizes an epitope which is masked when nesprin-1α is upregulated during osteoclast differentiation. DTT treatment restores epitope accessibility. Non-differentiated cells, indicated with an arrow, exhibit Sun2C reactivity. Scale bars: 10 µm. Images in A and E representative of three experimental repeats.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Derivative Assay, Functional Assay, Immunostaining, Expressing, RNA Expression, Staining

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: AKAP6 knockdown disrupts NE-MTOC formation in RAW-derived osteoclasts. (A) qPCR analysis of mRNA expression in siControl- and siAKAP6-treated osteoclasts. Bars represent mean fold change (normalised to siControl) for the indicated genes, with individual experimental values shown as dots (three independent experiments). Akap6 expression was significantly reduced following AKAP6 depletion, whereas Syne1a , Syne1 giant , the macrophage marker Emr1 , and the osteoclast markers Ctsk , Nfatc1 and Trap transcript levels were not significantly affected. ***P <0.001; ns, not significant (two-way ANOVA followed by Bonferroni's multiple comparisons test). (B–F) Immunostaining in RAW264.7-derived osteoclasts transfected with control siRNA (siControl) or AKAP6 siRNA (siAKAP6) of (B) AKAP6 (green) and GM130 (magenta), and DNA (DAPI); (C) nesprin-1 (green), CDK5RAP2 (magenta), and DNA (DAPI); (D) PCM1 (green), GM130 (magenta), and DNA (DAPI); (E) PCNT, (green), γ-tubulin (magenta), and DNA (DAPI); and (F) CDK5RAP2 (green), γ-tubulin (magenta), and DNA (DAPI). Note that although perinuclear localization of PCNT and CDK5RAP2 is lost upon AKAP6 knockdown, centrosomal localization is unaffected (arrows). Scale bars: 10 μm. Images in B–F representative of three experimental repeats.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Knockdown, Derivative Assay, Expressing, Marker, Immunostaining, Transfection, Control

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: AKAP6 Depletion disrupts NE-MTOC function, but not centrosomal-MTOC. (A) Immunostaining of α-tubulin (red), PCNT (green), GM130 (magenta) and DNA (DAPI) in RAW264.7-derived osteoclasts transfected with control siRNA (siControl) or AKAP6-targeting siRNA (siAKAP6). All stainings were performed in cells subjected to nocodazole washout (1.5 min). The yellow arrow indicates centrosomal microtubule organization and the arrowhead indicates Golgi-derived nucleation. (B) Quantification of fluorescence intensity profiles for α-tubulin, PCNT and GM130 in 0.2 µm wide concentric bands relative to distance from the nuclear edge (0.0). For each biological replicate ( n =3), 33–36 nuclei were analyzed per condition, and graphs show the mean±s.d. of the three independent biological experiments. The outward shift in the α-tubulin peak position is indicated by the red bracket, and the reduction in peak intensity is indicated by the green bracket. Statistical comparisons of peak position and peak intensity (amplitude) were performed at the experiment level with an unpaired two-tailed t -test; corresponding P -values are shown in the graphs. (C) Immunostaining of α-tubulin (red), EB1 (green), γ-tubulin (magenta) and DNA (DAPI) in RAW264.7-derived osteoclasts transfected with siControl or siAKAP6 performed after 1 min of nocodazole washout. Insets (2.5× magnification) highlight γ-tubulin-positive centrosomes exhibiting normal centrosomal microtubule outgrowth in both conditions. (D) Quantification of tubulin intensity in a 3 μm area surrounding each centrosome in siControl cells and siAKAP6 centrosomes from three independent experiments. SuperPlots display the overall mean (black line) and biological-replicate mean (color-coded triangles) from n =3, with the individual centrosomes (30–35 per condition) shown as matching color-coded dots superimposed beneath them. ns, not significant (paired two-tailed t -test). Scale bars: 10 μm.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Immunostaining, Derivative Assay, Transfection, Control, Fluorescence, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: AKAP6 depletion disrupts microtubule-actin cytoskeletal crosstalk in osteoclasts. (A) Representative images of RAW264.7-derived osteoclasts transfected with siControl or siAKAP6, stained for F-actin (phalloidin, green) and microtubules (α-tubulin, red). Corresponding BIOP JACoP analyses are shown in grayscale, illustrating the spatial overlap between F-actin and microtubules (lower panel). Scale bars: 10 μm. Dotted white lines indicate the boundary between the actin-dense sealing zone and the cytoplasm. (B) Quantification of microtubule-actin overlap in siControl and siAKAP6-treated osteoclasts. The actin-microtubule overlap was quantified in arbitrary units (a.u.) as the ratio of actin that colocalized with microtubules normalized against the total actin pixel count. SuperPlots display the overall mean (black line) and biological-replicate mean (color-coded triangles) from n =3, with the individual cells (12–25 per condition) shown as matching color-coded dots superimposed beneath them. ** P =0.0043 (paired two-tailed t -test). (C) Representative immunostaining images of RAW264.7-derived osteoclasts transfected with siControl or siAKAP6 co-stained for actin (phalloidin, green) and microtubules (α-tubulin, red). The right panels show higher magnification images of the boxed regions, highlighting actin ring structure. Images in C representative of three experimental repeats. Scale bars: 10 μm.

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Derivative Assay, Transfection, Staining, Two Tailed Test, Immunostaining

Journal: Journal of Cell Science

Article Title: Selective disruption of microtubule formation at the nuclear envelope impairs the bone resorption capacity of osteoclasts

doi: 10.1242/jcs.264166

Figure Lengend Snippet: AKAP6 depletion impairs osteoclast resorptive activity in RAW264.7-derived osteoclasts. (A) Representative images of RAW264.7-derived osteoclasts transfected with siControl- and siAKAP6 cultured on CaP-coated wells. Cells were removed, and the CaP substrate was stained with 2.5% AgNO 3 to visualize calcium deposits; resorption pits appear as unstained areas. Scale bars: 250 µm. (B) Relative resorbed area of siControl and siAKAP6-treated osteoclasts. Data represent mean±s.d. of three independent experiments, with three wells per experiment. **P =0.0081 (paired two-tailed t -test). (C) Representative images of RAW264.7-derived osteoclasts transfected with siControl or siAKAP6, and stained for F-actin (phalloidin, red), CaP (calcein, green) and nuclei (DAPI, blue). Note, calcein stains CaP, meaning resorption pits with lower calcein intensity indicate higher osteoclast resorption. Scale bars: 10 μm. (D) Quantification of osteoclast resorptive activity across sealing zone size categories (<500 μm 2 , 500–2000 μm 2 , >2000–5000 μm 2 and >5000 μm 2 ). SuperPlots display individual resorption pits as color-coded dots (siControl, blue; siAKAP6, red), with biological replicate means ( n =3; 35–80 pits per experiment, equal numbers per condition) superimposed as colored triangles, each color representing a different replicate. Error bars show overall mean±s.d. Resorption was quantified using the calcein ratio (calcein intensity inside the sealing zone divided by the intensity outside), where values <1 indicate active resorption. *P <0.05; **P <0.01 (two-way ANOVA with Bonferroni's post-hoc test).

Article Snippet: RAW264.7 cells were obtained from ATCC and maintained in high glucose DMEM supplemented with GlutaMAX (Thermo Fisher Scientific, 61965059) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, 26140079), 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, 15140-122) and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Techniques: Activity Assay, Derivative Assay, Transfection, Cell Culture, Staining, Two Tailed Test