culture medium  (PromoCell)

Journal: Bioactive Materials

Article Title: Injectable microgels loaded with programmable WELNs based on mendelian randomization macroanalysis alleviate osteoarthritis by restoring the circadian rhythm

doi: 10.1016/j.bioactmat.2025.10.032

Figure Lengend Snippet: Biosafety, cellular uptake, protected mitochondria and promoted ECM synthesis of WELNs-CAP. (a) Cellular uptake of WELNs-CAP in chondrocytes. Nuclei stained with DAPI, WELNs and WELNs-CAP labeled with PKH26 and the membrane of chondrocytes marked with DiO. Scale bar = 50 μm. (b) Statistical plot of red fluorescence intensity of WELNs and WELNs-CAP. (c) JC-1 staining of chondrocytes after different treatments. Aggregation exhibited red fluorescence (UCF 590 ), monomer exhibited green fluorescence (UCF 530 ). Scale bar = 50 μm. (d) Analysis of fluorescence ratio of UCF 590 /UCF 530 . (e) Cytoskeleton staining. Nuclei stained with DAPI, β-ACTIN stained with phalloidin. Scale bar = 10 μm. (f) The length/width ratio of chondrocytes under different treatments. (g) Detected the mRNA expression levels of genes related to catabolism, anabolism and inflammatory factors ( Mmp13 , Adamts5 , Aggrecan , Col2a1 , Inos and Il-1β ) using Real-time quantitative PCR (RT-qPCR), displayed by heat map. Red represented high expression level, blue represented low expression level. (h) Western blot bands of ADAMTS5, SOX9, MMP13, and GAPDH in chondrocytes under different treatments. (i–k) Semi-quantitative statistics of ADAMTS5, SOX9 and MMP13 proteins. The data of each group was the normalized data, which the target gene expression relative to the Ctrl group. (l) Immunofluorescence staining of COLⅡ and MMP13 in chondrocytes performed under different treatments. Scale bar = 50 μm. (m, n) Semi-quantitative statistics of COLII and MMP13 proteins. n = 3 in all experiments. The data of each group was the normalized data, which the target gene expression relative to the Ctrl group. Statistical analysis was performed using two-tailed unpaired t -test for b and one-way ANOVA for (f, i, j, k, m and n), ns is no significant different with p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Fixed chondrocytes and sections which obtained from articular cartilage and explants were permeabilized with Triton X-100 (Beyotime) for 8 min. After incubating with blocking buffer (3 % BSA solution), the sections and chondrocytes were incubated with primary antibodies including anti-MMP13 (1:100, 18165-1-AP, Proteintech; 1:150, ab39012, Abcam), anti- Collagen II (1:100, 28459-1-AP, Proteintech; 1:100, ab34712, Abcam), anti-BMAL1 (1:100, ab230822, Abcam) and anti-KLF10 (1:25, bs-1838R, Bioss) overnight at 4 °C and then washed.

Techniques: Staining, Labeling, Membrane, Fluorescence, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Targeted Gene Expression, Immunofluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: Injectable microgels loaded with programmable WELNs based on mendelian randomization macroanalysis alleviate osteoarthritis by restoring the circadian rhythm

doi: 10.1016/j.bioactmat.2025.10.032

Figure Lengend Snippet: FA and KLF10-IN-1 regulated the circadian rhythm by binding to KLF10. (a, c, e) Rhythm fitting curves of Bmal1 , Clock and Per2 within 48 h in Ctrl, IL-1β, IL-1β+FA and IL-1β+KLF10-IN-1 groups in primary chondrocytes. (b, d, f) Quantification of the Bmal1 , Clock and Per2 amplitude, phase and mesor in a, c and e, respectively. (g, i, k) Rhythm fitting curves of BMAL1 , CLOCK and PER2 within 48 h in Vector, OE, OE + FA and OE + KLF10-IN-1 groups in SW1353 cells. (h, j, l) Quantification of the BMAL1 , CLOCK and PER2 amplitude, phase and mesor in g, i and k, respectively. The results of RT-PCR were analyzed by the circa compare algorithm. The expression of mRNA was relative-quantified using the 2 −ΔΔCt algorithm. RStudio software was used to perform 24-h cosine rhythmicity analysis of BMAL1 , CLOCK and PER2 genes expression, calculate three key rhythmic parameters—amplitude, phase, and mesor, and subjected these parameters to rigorous statistical validation. Statistical analysis was performed using two-tailed unpaired t -test for n and one-way ANOVA for (b, d, f, h, j, l and p), ns is no significant different with p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Fixed chondrocytes and sections which obtained from articular cartilage and explants were permeabilized with Triton X-100 (Beyotime) for 8 min. After incubating with blocking buffer (3 % BSA solution), the sections and chondrocytes were incubated with primary antibodies including anti-MMP13 (1:100, 18165-1-AP, Proteintech; 1:150, ab39012, Abcam), anti- Collagen II (1:100, 28459-1-AP, Proteintech; 1:100, ab34712, Abcam), anti-BMAL1 (1:100, ab230822, Abcam) and anti-KLF10 (1:25, bs-1838R, Bioss) overnight at 4 °C and then washed.

Techniques: Binding Assay, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Expressing, Software, Biomarker Discovery, Two Tailed Test

Journal: Bioactive Materials

Article Title: Injectable microgels loaded with programmable WELNs based on mendelian randomization macroanalysis alleviate osteoarthritis by restoring the circadian rhythm

doi: 10.1016/j.bioactmat.2025.10.032

Figure Lengend Snippet: FA and KLF10-IN-1 regulated the circadian rhythm by binding to KLF10 to alleviate OA in vitro. (a) Western blot bands of BMAL1, SOX9, MMP13, and β-ACTIN in Ctrl, IL-1β and IL-1β+KLF10-IN-1 groups in primary chondrocytes. (b) Semi-quantitative statistics of BMAL1, SOX9 and MMP13 proteins. (c) Immunofluorescence staining of BMAL1 in primary chondrocytes performed in Ctrl, IL-1β and IL-1β+KLF10-IN-1 groups. (d) Semi-quantitative statistics of BMAL1 proteins. (e) SF staining on sections of FHCs in Ctrl, IL-1β and IL-1β+KLF10-IN-1 groups, and ROI were marked using black dashed lines. (f) Area fraction analysis of fast green stained in the ROI. (g) Western blot bands of BMAL1, SOX9, MMP13, and β-ACTIN in chondrocytes under different treatments. (h) Semi-quantitative statistics of BMAL1, SOX9 and MMP13 proteins. (i) Immunofluorescence staining of COLⅡ, MMP13 and BMAL1 in chondrocytes performed in Ctrl, IL-1β and IL-1β+FA groups. (j) Semi-quantitative statistics of COLⅡ, MMP13 and BMAL1 proteins. (k) SF staining on sections of FHCs in Ctrl, IL-1β and IL-1β+FA groups, and ROI were marked using black dashed lines. (l) Area fraction analysis of fast green stained in the ROI. (m) Immunofluorescence staining of COLⅡ and MMP13 on sections of FHCs performed under different treatments. (n) Semi-quantitative statistics of COLⅡ and MMP13 proteins. (o) Western blot bands of SOX9 and MMP13 in SW1353 cells under different treatments. (p) Semi-quantitative statistics of SOX9 and MMP13 proteins in (o). (q) Immunofluorescence staining of COLⅡ and MMP13 in Vector, OE, OE + FA and OE + KLF10-IN-1 groups in SW1353 cells. Scale bar = 50 μm. (r) Semi-quantitative statistics of COLⅡ and MMP13 proteins, the data of each group were the normalized data. n = 3 in all experiments and the data of each group were the normalized data, which the target gene expression relative to the Ctrl group expect (f, l and r). Statistical analysis was performed using one-way ANOVA, ns is no significant different with p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Fixed chondrocytes and sections which obtained from articular cartilage and explants were permeabilized with Triton X-100 (Beyotime) for 8 min. After incubating with blocking buffer (3 % BSA solution), the sections and chondrocytes were incubated with primary antibodies including anti-MMP13 (1:100, 18165-1-AP, Proteintech; 1:150, ab39012, Abcam), anti- Collagen II (1:100, 28459-1-AP, Proteintech; 1:100, ab34712, Abcam), anti-BMAL1 (1:100, ab230822, Abcam) and anti-KLF10 (1:25, bs-1838R, Bioss) overnight at 4 °C and then washed.

Techniques: Binding Assay, In Vitro, Western Blot, Immunofluorescence, Staining, Plasmid Preparation, Targeted Gene Expression