Journal: Molecular Medicine Reports

Article Title: Leptin promotes obesity-related osteoarthritis progression via CD44/JAK2/STAT3 axis-mediated chondrocyte apoptosis

doi: 10.3892/mmr.2026.14039

Figure Lengend Snippet: Leptin expression is significantly elevated in obesity-related OA. (A) H&E staining and Safranin O-fast green staining showed cartilage surface fibrillation/tearing, cartilage thinning, and chondrocyte disorganization in both groups. (B) OARSI scores showed no significant difference between groups. IHC showed significantly more leptin-positive cells in obesity-related OA than in non-obesity-related OA. Black boxes in mark magnified regions; lower panels show high-magnification views. Scale bar, 100 µm. ****P<0.0001. ns, not significant; OA, osteoarthritis; OARSI, Osteoarthritis Research Society International.

Article Snippet: The antibodies used were as follows: BAX (dilution, 1:2,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), BCL-2 (dilution, 1:1,000; cat. no. T40056 ; Abmart Pharmaceutical Technology Co., Ltd.), caspase-3 (dilution, 1:3,000; cat. no. 19677-1-AP; Proteintech Group, Inc.), cleaved caspase-3 (dilution, 1:3,000; cat. no. 25128-1-AP; Proteintech Group, Inc.), CD44 (dilution, 1:2,000; cat. no. 30854-1-AP; Proteintech Group, Inc.), JAK2 (dilution, 1:1,000; cat. no. T55287 ; Abmart Pharmaceutical Technology Co., Ltd.), p-JAK2 (dilution, 1:500; cat. no. T56570 ; Abmart Pharmaceutical Technology Co., Ltd.), STAT3 (dilution, 1:2,000; cat. no. 10253-2-AP; Proteintech Group, Inc.), p-STAT3 (dilution, 1:1,000; cat. no. 28945-1-AP; Proteintech Group, Inc.), leptin receptor isoform B (Ob-Rb; dilution, 1:500; cat. no. 20966-1-AP; Proteintech Group, Inc.) and β-actin (dilution, 1:4,000; cat. no. 20536-1-AP; Proteintech Group, Inc.).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Molecular Medicine Reports

Article Title: Leptin promotes obesity-related osteoarthritis progression via CD44/JAK2/STAT3 axis-mediated chondrocyte apoptosis

doi: 10.3892/mmr.2026.14039

Figure Lengend Snippet: DEGs and enrichment analysis results. (A) Volcano plot showing differentially expressed genes between the PBS-treated and leptin-treated groups. (B) GSEA enrichment of the JAK2/STAT3 signaling pathway in published transcriptomics data from GSE85785 . (C) GSEA enrichment of apoptosis in published transcriptomics data from GSE85785 . (D) Venn diagram displaying the determination of 4 key genes (CD38, CD44, IL-6 and IL-1β). DEGS, differentially expressed genes; JAK2, Janus kinase 2; GSEA, gene set enrichment analysis; NES, normalized enrichment score.

Article Snippet: The antibodies used were as follows: BAX (dilution, 1:2,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), BCL-2 (dilution, 1:1,000; cat. no. T40056 ; Abmart Pharmaceutical Technology Co., Ltd.), caspase-3 (dilution, 1:3,000; cat. no. 19677-1-AP; Proteintech Group, Inc.), cleaved caspase-3 (dilution, 1:3,000; cat. no. 25128-1-AP; Proteintech Group, Inc.), CD44 (dilution, 1:2,000; cat. no. 30854-1-AP; Proteintech Group, Inc.), JAK2 (dilution, 1:1,000; cat. no. T55287 ; Abmart Pharmaceutical Technology Co., Ltd.), p-JAK2 (dilution, 1:500; cat. no. T56570 ; Abmart Pharmaceutical Technology Co., Ltd.), STAT3 (dilution, 1:2,000; cat. no. 10253-2-AP; Proteintech Group, Inc.), p-STAT3 (dilution, 1:1,000; cat. no. 28945-1-AP; Proteintech Group, Inc.), leptin receptor isoform B (Ob-Rb; dilution, 1:500; cat. no. 20966-1-AP; Proteintech Group, Inc.) and β-actin (dilution, 1:4,000; cat. no. 20536-1-AP; Proteintech Group, Inc.).

Techniques: Analysis, Transcriptomics, Significance Assay

Journal: Molecular Medicine Reports

Article Title: Leptin promotes obesity-related osteoarthritis progression via CD44/JAK2/STAT3 axis-mediated chondrocyte apoptosis

doi: 10.3892/mmr.2026.14039

Figure Lengend Snippet: Screening for leptin concentration and identification of key genes. (A) Cell Counting Kit-8 assay results demonstrated that the IC 50 of leptin was 85.05 ng/ml. (B) RT-qPCR quantification indicated a significant increase in CD44 expression in C28/I2 chondrocytes upon leptin stimulation (80 ng/ml). (C) RT-qPCR analysis of CD44 expression at various leptin concentrations. (D) Western blotting and (E) semi-quantification of CD44 protein expression at various leptin concentrations. **P<0.01 and ****P<0.0001. ns, not significant. RT-qPCR, Reverse transcription-quantitative PCR.

Article Snippet: The antibodies used were as follows: BAX (dilution, 1:2,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), BCL-2 (dilution, 1:1,000; cat. no. T40056 ; Abmart Pharmaceutical Technology Co., Ltd.), caspase-3 (dilution, 1:3,000; cat. no. 19677-1-AP; Proteintech Group, Inc.), cleaved caspase-3 (dilution, 1:3,000; cat. no. 25128-1-AP; Proteintech Group, Inc.), CD44 (dilution, 1:2,000; cat. no. 30854-1-AP; Proteintech Group, Inc.), JAK2 (dilution, 1:1,000; cat. no. T55287 ; Abmart Pharmaceutical Technology Co., Ltd.), p-JAK2 (dilution, 1:500; cat. no. T56570 ; Abmart Pharmaceutical Technology Co., Ltd.), STAT3 (dilution, 1:2,000; cat. no. 10253-2-AP; Proteintech Group, Inc.), p-STAT3 (dilution, 1:1,000; cat. no. 28945-1-AP; Proteintech Group, Inc.), leptin receptor isoform B (Ob-Rb; dilution, 1:500; cat. no. 20966-1-AP; Proteintech Group, Inc.) and β-actin (dilution, 1:4,000; cat. no. 20536-1-AP; Proteintech Group, Inc.).

Techniques: Screening, Concentration Assay, Cell Counting, Quantitative RT-PCR, Expressing, Analysis, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Leptin promotes obesity-related osteoarthritis progression via CD44/JAK2/STAT3 axis-mediated chondrocyte apoptosis

doi: 10.3892/mmr.2026.14039

Figure Lengend Snippet: Leptin promotes CD44 expression, activates the JAK2/STAT3 signaling pathway and induces apoptosis in C28/I2 chondrocytes. (A) Western blotting and (B) semi-quantification of CD44 in C28/I2 chondrocytes upon leptin stimulation. Β-actin served as a loading control. (C) Reverse transcription-quantitative PCR analysis of changes in BAX, BCL-2 and caspase-3 mRNA abundance upon leptin stimulation. (D) Quantification of CD44 upon leptin stimulation and (E) immunofluorescence detection (scale bar, 100 µM). (F) Western blotting and (G) semi-quantification of apoptosis-related proteins (BAX, BCL-2, caspase-3 and cleaved caspase-3) upon leptin stimulation. (H) Quantification of apoptosis upon leptin stimulation and (I) TUNEL staining detection (scale bar, 50 µm). (K) Flow cytometric assessment of apoptotic rate and (L) quantification of apoptosis upon leptin stimulation. (J) Western blotting and (M) semi-quantification of p-JAK2, JAK2, p-STAT3 and STAT3 upon leptin stimulation. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. JAK2, Janus kinase 2; p-, phosphorylated.

Article Snippet: The antibodies used were as follows: BAX (dilution, 1:2,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), BCL-2 (dilution, 1:1,000; cat. no. T40056 ; Abmart Pharmaceutical Technology Co., Ltd.), caspase-3 (dilution, 1:3,000; cat. no. 19677-1-AP; Proteintech Group, Inc.), cleaved caspase-3 (dilution, 1:3,000; cat. no. 25128-1-AP; Proteintech Group, Inc.), CD44 (dilution, 1:2,000; cat. no. 30854-1-AP; Proteintech Group, Inc.), JAK2 (dilution, 1:1,000; cat. no. T55287 ; Abmart Pharmaceutical Technology Co., Ltd.), p-JAK2 (dilution, 1:500; cat. no. T56570 ; Abmart Pharmaceutical Technology Co., Ltd.), STAT3 (dilution, 1:2,000; cat. no. 10253-2-AP; Proteintech Group, Inc.), p-STAT3 (dilution, 1:1,000; cat. no. 28945-1-AP; Proteintech Group, Inc.), leptin receptor isoform B (Ob-Rb; dilution, 1:500; cat. no. 20966-1-AP; Proteintech Group, Inc.) and β-actin (dilution, 1:4,000; cat. no. 20536-1-AP; Proteintech Group, Inc.).

Techniques: Expressing, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Analysis, Immunofluorescence, TUNEL Assay, Staining

Journal: Molecular Medicine Reports

Article Title: Leptin promotes obesity-related osteoarthritis progression via CD44/JAK2/STAT3 axis-mediated chondrocyte apoptosis

doi: 10.3892/mmr.2026.14039

Figure Lengend Snippet: Leptin induces apoptosis, which is attenuated by the JAK2 inhibitor AG490. (A) Western blotting and (B) semi-quantification of apoptosis-related proteins (BAX, BCL-2, caspase-3 and cleaved caspase-3) in C28/I2 chondrocytes treated with leptin and either with or without AG490. (C) TUNEL staining and (D) quantification of apoptosis in C28/I2 chondrocytes treated with leptin in the presence or absence of AG490. (scale bar, 100 µm). (E) Flow cytometric detection of apoptosis in C28/I2 chondrocytes treated with PBS, leptin, or leptin + AG490. (F) Quantification of the apoptotic rate. *P<0.05, ***P<0.001, and ****P<0.0001. ns, not significant.

Article Snippet: The antibodies used were as follows: BAX (dilution, 1:2,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), BCL-2 (dilution, 1:1,000; cat. no. T40056 ; Abmart Pharmaceutical Technology Co., Ltd.), caspase-3 (dilution, 1:3,000; cat. no. 19677-1-AP; Proteintech Group, Inc.), cleaved caspase-3 (dilution, 1:3,000; cat. no. 25128-1-AP; Proteintech Group, Inc.), CD44 (dilution, 1:2,000; cat. no. 30854-1-AP; Proteintech Group, Inc.), JAK2 (dilution, 1:1,000; cat. no. T55287 ; Abmart Pharmaceutical Technology Co., Ltd.), p-JAK2 (dilution, 1:500; cat. no. T56570 ; Abmart Pharmaceutical Technology Co., Ltd.), STAT3 (dilution, 1:2,000; cat. no. 10253-2-AP; Proteintech Group, Inc.), p-STAT3 (dilution, 1:1,000; cat. no. 28945-1-AP; Proteintech Group, Inc.), leptin receptor isoform B (Ob-Rb; dilution, 1:500; cat. no. 20966-1-AP; Proteintech Group, Inc.) and β-actin (dilution, 1:4,000; cat. no. 20536-1-AP; Proteintech Group, Inc.).

Techniques: Western Blot, TUNEL Assay, Staining

Journal: Molecular Medicine Reports

Article Title: Leptin promotes obesity-related osteoarthritis progression via CD44/JAK2/STAT3 axis-mediated chondrocyte apoptosis

doi: 10.3892/mmr.2026.14039

Figure Lengend Snippet: Leptin promotes chondrocyte apoptosis by stimulating the JAK2/STAT3 signaling pathway through CD44. (A) Western blotting and (B) semi-quantification of apoptosis-related proteins (BAX, BCL-2, caspase-3 and cleaved caspase-3) in C28/I2 chondrocytes from PBS and leptin groups following CD44 knockdown. (C) Flow cytometric detection of apoptosis in C28/I2 chondrocytes treated with PBS, sh-CD44, leptin, or leptin + sh-CD44. (D) Quantification of the apoptotic rate. (E) Western blotting and (F) semi-quantification of JAK2/STAT3 pathway-related proteins (p-JAK2, JAK2, p-STAT3 and STAT3) following CD44 knockdown. (G) Immunofluorescence examination of STAT3 nuclear translocation in the leptin-treated group following CD44 knockdown (scale bar, 50 µm). **P<0.01, ***P<0.001 and ****P<0.0001. JAK2, Janus kinase 2; ns, not significant; p-, phosphorylated.

Article Snippet: The antibodies used were as follows: BAX (dilution, 1:2,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), BCL-2 (dilution, 1:1,000; cat. no. T40056 ; Abmart Pharmaceutical Technology Co., Ltd.), caspase-3 (dilution, 1:3,000; cat. no. 19677-1-AP; Proteintech Group, Inc.), cleaved caspase-3 (dilution, 1:3,000; cat. no. 25128-1-AP; Proteintech Group, Inc.), CD44 (dilution, 1:2,000; cat. no. 30854-1-AP; Proteintech Group, Inc.), JAK2 (dilution, 1:1,000; cat. no. T55287 ; Abmart Pharmaceutical Technology Co., Ltd.), p-JAK2 (dilution, 1:500; cat. no. T56570 ; Abmart Pharmaceutical Technology Co., Ltd.), STAT3 (dilution, 1:2,000; cat. no. 10253-2-AP; Proteintech Group, Inc.), p-STAT3 (dilution, 1:1,000; cat. no. 28945-1-AP; Proteintech Group, Inc.), leptin receptor isoform B (Ob-Rb; dilution, 1:500; cat. no. 20966-1-AP; Proteintech Group, Inc.) and β-actin (dilution, 1:4,000; cat. no. 20536-1-AP; Proteintech Group, Inc.).

Techniques: Western Blot, Knockdown, Immunofluorescence, Translocation Assay

Journal: Molecular Medicine Reports

Article Title: Leptin promotes obesity-related osteoarthritis progression via CD44/JAK2/STAT3 axis-mediated chondrocyte apoptosis

doi: 10.3892/mmr.2026.14039

Figure Lengend Snippet: CD44 overexpression exacerbates leptin-induced chondrocyte apoptosis and JAK2/STAT3 activation. (A) Western blotting and (B) semi-quantification of apoptosis-related proteins (BAX, BCL-2, caspase-3 and cleaved caspase-3) in C28/I2 chondrocytes from PBS and leptin groups following CD44 overexpression. (C) Flow cytometric detection of apoptosis in C28/I2 chondrocytes treated with PBS, OE-CD44, leptin, or leptin + OE-CD44. (D) Quantification of the apoptotic rate. (E) Western blotting and (F) semi-quantification of JAK2/STAT3 pathway-related proteins (p-JAK2, JAK2, p-STAT3 and STAT3) following CD44 overexpression. (G) Western blotting analysis of Ob-Rb protein expression following CD44 knockdown or overexpression. (H) Quantification of Ob-Rb protein expression from the Western blotting. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. JAK2, Janus kinase 2; ns, not significant; Ob-Rb, leptin receptor isoform B; p-, phosphorylated.

Article Snippet: The antibodies used were as follows: BAX (dilution, 1:2,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), BCL-2 (dilution, 1:1,000; cat. no. T40056 ; Abmart Pharmaceutical Technology Co., Ltd.), caspase-3 (dilution, 1:3,000; cat. no. 19677-1-AP; Proteintech Group, Inc.), cleaved caspase-3 (dilution, 1:3,000; cat. no. 25128-1-AP; Proteintech Group, Inc.), CD44 (dilution, 1:2,000; cat. no. 30854-1-AP; Proteintech Group, Inc.), JAK2 (dilution, 1:1,000; cat. no. T55287 ; Abmart Pharmaceutical Technology Co., Ltd.), p-JAK2 (dilution, 1:500; cat. no. T56570 ; Abmart Pharmaceutical Technology Co., Ltd.), STAT3 (dilution, 1:2,000; cat. no. 10253-2-AP; Proteintech Group, Inc.), p-STAT3 (dilution, 1:1,000; cat. no. 28945-1-AP; Proteintech Group, Inc.), leptin receptor isoform B (Ob-Rb; dilution, 1:500; cat. no. 20966-1-AP; Proteintech Group, Inc.) and β-actin (dilution, 1:4,000; cat. no. 20536-1-AP; Proteintech Group, Inc.).

Techniques: Over Expression, Activation Assay, Western Blot, Analysis, Expressing, Knockdown

Journal: Molecular Medicine Reports

Article Title: Leptin promotes obesity-related osteoarthritis progression via CD44/JAK2/STAT3 axis-mediated chondrocyte apoptosis

doi: 10.3892/mmr.2026.14039

Figure Lengend Snippet: Leptin promotes CD44 expression, activates the JAK2/STAT3 signaling pathway, induces chondrocyte apoptosis and exacerbates osteoarthritis in vivo . (A) Western blotting and (B) semi-quantification of JAK2/STAT3 pathway-related proteins (p-JAK2, JAK2, p-STAT3 and STAT3) in rat chondrocytes. (C) H&E and Safranin O-Fast Green staining images of articular cartilage, subchondral bone and synovium, as well as IHC staining for CD44 in articular cartilage across the four groups. These images show cartilage degeneration, characterized by surface fibrillation, cartilage thinning, chondrocyte disorganization, reduced Safranin O staining (indicating proteoglycan loss) and subchondral bone remodeling and synovial hyperplasia. The black rectangular boxes mark the magnified regions, and the lower panels show high-magnification views (scale bar, 200 µm). (D) Quantification of cartilage degeneration, subchondral bone remodeling, synovium inflammation/hyperplasia and the percentage of CD44-positive cells in articular cartilage across the four groups. (E) TUNEL staining detection and (F) quantification of apoptosis in rat cartilage tissue (scale bar, 100 µm). ****P<0.0001. JAK2, Janus kinase 2; ns, not significant; shRNA, short hairpin RNA; p-, phosphorylated.

Article Snippet: The antibodies used were as follows: BAX (dilution, 1:2,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), BCL-2 (dilution, 1:1,000; cat. no. T40056 ; Abmart Pharmaceutical Technology Co., Ltd.), caspase-3 (dilution, 1:3,000; cat. no. 19677-1-AP; Proteintech Group, Inc.), cleaved caspase-3 (dilution, 1:3,000; cat. no. 25128-1-AP; Proteintech Group, Inc.), CD44 (dilution, 1:2,000; cat. no. 30854-1-AP; Proteintech Group, Inc.), JAK2 (dilution, 1:1,000; cat. no. T55287 ; Abmart Pharmaceutical Technology Co., Ltd.), p-JAK2 (dilution, 1:500; cat. no. T56570 ; Abmart Pharmaceutical Technology Co., Ltd.), STAT3 (dilution, 1:2,000; cat. no. 10253-2-AP; Proteintech Group, Inc.), p-STAT3 (dilution, 1:1,000; cat. no. 28945-1-AP; Proteintech Group, Inc.), leptin receptor isoform B (Ob-Rb; dilution, 1:500; cat. no. 20966-1-AP; Proteintech Group, Inc.) and β-actin (dilution, 1:4,000; cat. no. 20536-1-AP; Proteintech Group, Inc.).

Techniques: Expressing, In Vivo, Western Blot, Staining, Immunohistochemistry, TUNEL Assay, Tissue, shRNA

Journal: Molecular Medicine Reports

Article Title: Leptin promotes obesity-related osteoarthritis progression via CD44/JAK2/STAT3 axis-mediated chondrocyte apoptosis

doi: 10.3892/mmr.2026.14039

Figure Lengend Snippet: A model of the mechanism of leptin in osteoarthritis. Following leptin stimulation, CD44 expression increases, activating the JAK2/STAT3 signaling pathway. Overactivation of this pathway induces chondrocyte apoptosis, ultimately promoting osteoarthritis progression. Casp3, caspase-3; c-Casp3, cleaved caspase-3; JAK2, Janus kinase 2.

Article Snippet: The antibodies used were as follows: BAX (dilution, 1:2,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), BCL-2 (dilution, 1:1,000; cat. no. T40056 ; Abmart Pharmaceutical Technology Co., Ltd.), caspase-3 (dilution, 1:3,000; cat. no. 19677-1-AP; Proteintech Group, Inc.), cleaved caspase-3 (dilution, 1:3,000; cat. no. 25128-1-AP; Proteintech Group, Inc.), CD44 (dilution, 1:2,000; cat. no. 30854-1-AP; Proteintech Group, Inc.), JAK2 (dilution, 1:1,000; cat. no. T55287 ; Abmart Pharmaceutical Technology Co., Ltd.), p-JAK2 (dilution, 1:500; cat. no. T56570 ; Abmart Pharmaceutical Technology Co., Ltd.), STAT3 (dilution, 1:2,000; cat. no. 10253-2-AP; Proteintech Group, Inc.), p-STAT3 (dilution, 1:1,000; cat. no. 28945-1-AP; Proteintech Group, Inc.), leptin receptor isoform B (Ob-Rb; dilution, 1:500; cat. no. 20966-1-AP; Proteintech Group, Inc.) and β-actin (dilution, 1:4,000; cat. no. 20536-1-AP; Proteintech Group, Inc.).

Techniques: Expressing

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS attenuates full-blown bone marrow senescence during GC-induced skeletal degeneration. ( A ) Schematic illustration of the experimental design for assessing bone marrow senescence at 4 weeks after combined SCS and MPS treatment. ( B ) Representative images of SA-β-Gal–positive cells (green) in femur after MPS treatment. BM indicates bone marrow; TBM indicates trabecular bone matrix. (Scale bars, 100 μm and 25 μm) ( C – E ) Representative immunofluorescence images at week 4 showing Emcn + sinusoidal ECs, ALP + osteoblasts, and p16 + senescent cells (C), with corresponding quantification of Emcn + p16 + (D) and ALP + p16 + cells (E). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) ( F – H ) Flow cytometry analysis of CD45 − Ter119 − CD31 + arteriolar ECs in the femur after PBS or SCS treatment (F). Ki-67 + proliferative status was further analyzed within this population (G), and corresponding double-positive cell quantification is shown in (H). n = 6 biological replicates. ( I – K ) Representative flow cytometry plots of CD45 − Ter119 − CD31 − leptin receptor + (LepR + ) mesenchymal stem cells (MSCs) in the bone marrow at 4 weeks (I), with analysis of the proportion of SA-β-Gal–positive cells (J) and corresponding quantification (K). n = 6 biological replicates. ( L ) Representative flow cytometry plots of CD45 − Ter119 − CD144 + cells (including endothelial cells and endothelial progenitors) in the bone marrow at week 4 post-MPS treatment. ( M and N ) Gating and analysis of CD45 − Ter119 − CD144 + HMGB1 + ECs by flow cytometry (M), and corresponding quantification (N). n = 6 biological replicates. ( O and P ) Representative immunofluorescence images showing OPN + osteoblasts and γ-H2A.X + DNA damage marker–positive cells in the femur at 4 weeks (O), with quantification of senescent osteoblasts (P). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined using an unpaired two-tailed Student's t -test ( D, E, H, K, N and P ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Immunofluorescence, Flow Cytometry, Marker, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS reprograms the lineage commitment of MSCs after GC treatment and inhibits the generation of primary senescent adipocytes. ( A ) Schematic illustration of the in vitro investigation of SCS targeting the prostaglandin/PPARγ/INK positive feedback loop in MPS-induced primary senescent adipocytes. ( B ) Representative flow cytometry plot showing p16 + senescent cells in adipocytes derived from bone marrow after 14 days of in vivo MPS induction and subsequently treated with SCS in vitro . ( C ) qPCR analysis of 12 senescence-associated markers in primary senescent adipocytes after in vitro SCS treatment. n = 3 biological replicates. ( D ) ELISA analysis of IL-1β levels in adipocyte supernatant following in vitro SCS treatment. n = 6 biological replicates. ( E ) ELISA analysis of secreted prostaglandins PGD2 and PGE2 in adipocytes under different treatment conditions. D-PBS: bone marrow adipocytes isolated from mice treated in vivo with the solvent control DMSO, followed by in vitro treatment with PBS; M-PBS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with PBS. M-SCS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with SCS. ( F ) Western blot analysis of intracellular COX-2 protein levels in adipocytes across the three treatment conditions. ( G ) Schematic illustration of competitive osteogenic–adipogenic differentiation of CD45 − Ter119 − CD31 − LepR + MSCs after 7 days of in vivo SCS and MPS co-treatment. ( H ) qPCR analysis of pan-adipocyte markers ( Fabp4 , Adipoq , Plin1 , Cd36 , and Lep ) in CD45 − Ter119 − CD31 − LepR + MSCs after 14 days of in vitro competitive lineage differentiation. n = 3 biological replicates. ( I and J ) Representative immunofluorescence images (I) and quantification (J) of perilipin + adipocytes and osteopontin + mature osteoblasts derived from lineage-committed MSCs. n = 6 biological replicates. (Scale bars, 30 μm, 15 μm and 15 μm). ( K ) Western blot analysis of adipogenesis-related markers C/EBPα, PPARγ, and C/EBPβ in the lineage-mixed cells after in vitro competitive differentiation of CD45 − Ter119 − CD31 − LepR + MSCs. ( L ) qPCR analysis of lipogenesis-related markers Fasn , Scd1 , Srebf1 , Acaca , and Acacb . n = 3 biological replicates. ( M and N ) Representative H&E staining images (M) of the femurs at day 14 following SCS and MPS co-treatment. Yellow arrows indicate bone marrow adipocytes. Magnified images show hypertrophic adipocyte morphology, with quantification of adipocyte diameter (N). n = 19 biological replicates. (Scale bars, 200 μm, 50 μm and 20 μm). Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( C, D, H, J, L and N ), or one-way ANOVA with Tukey's post hoc test ( E ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: In Vitro, Flow Cytometry, Derivative Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Solvent, Control, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Gene expression profiles of bone marrow-derived LepR + MSCs after 7-day in vivo co-treatment with SCS and MPS. ( A ) Heatmap showing DEGs in CD45 − Ter119 − CD31 − LepR + MSCs sorted from bone marrow at day 7 post-treatment with SCS versus PBS ( P < 0.05, |log fold change| > 1.5). n = 3 biological replicates. ( B ) Representative GO biological process enrichment analysis of downregulated DEGs. ( C ) Top 20 enriched KEGG pathways of downregulated DEGs in SCS versus PBS. ( D ) GSEA plots of biological processes positively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25). ( E ) Representative downregulated DEGs associated with adipogenesis and lipogenesis identified through KEGG pathway analysis. n = 3 biological replicates. ( F ) Top 20 enriched KEGG pathways of upregulated DEGs in SCS versus PBS. ( G ) Representative GO biological process enrichment analysis of upregulated DEGs. ( H ) Representative upregulated DEGs identified through biological process enrichment analysis. n = 3 biological replicates. ( I and J ) GSEA plots of KEGG pathways negatively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Gene Expression, Derivative Assay, In Vivo

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

Journal: Biomaterials Research

Article Title: Oxidative Stress and Hormone-Regulated Dermal Papilla Cell-Targeted Nanomodulators: Reverse Cellular Senescence for Androgenetic Alopecia Therapy

doi: 10.34133/bmr.0333

Figure Lengend Snippet: Schematic illustration of the fabrication process (A) and therapeutic AGA mechanism (B) of DPC-targeted nanocarriers (L-LP-Fi/CeNPs) that synergistically inhibit DPC senescence. DPC, dermal papilla cell; PEG, polyethylene glycol; CeNP, cerium oxide nanoparticle; Fi, finasteride; L-LP, leptin-functionalized, co-loaded liposome; DP, dermal papilla; DHT, dihydrotestosterone; ROS, reactive oxygen species; HFSC, hair follicle stem cell; TGF-β, transforming growth factor β; AGA, androgenetic alopecia; LEPR, leptin receptor; T, testosterone; SOD, superoxide dismutase; CAT, catalase.

Article Snippet: Intracellular LEPR was labeled with a rabbit anti-LEPR protein monoclonal antibody (1:100, HUABIO), followed by an Alexa Fluor 561 goat anti-rabbit secondary antibody incubation.

Techniques:

Journal: Biomaterials Research

Article Title: Oxidative Stress and Hormone-Regulated Dermal Papilla Cell-Targeted Nanomodulators: Reverse Cellular Senescence for Androgenetic Alopecia Therapy

doi: 10.34133/bmr.0333

Figure Lengend Snippet: LEPR expression in DPCs and validation of binding to leptin. (A) LEPR expression in HDPCs, HaCaT cells, MDPCs, and mEK cells. Scale bar = 50 μm. (B) Quantitative analysis of LEPR expression in cells ( n = 3). (C) Time scale analysis of the HF cycle in C57BL/6 mice at 11 weeks after birth. (D) LEPR expression in different HF phases of mice. Scale bar = 25 μm. (E and F) Western blotting and grayscale analysis of LEPR expression (HDPC and HaCaT) ( n = 3). (G) The mechanism by which leptin targets LEPR-expressing DPCs. (H) CLSM imaging following co-incubation with FITC-labeled leptin (green) and Alexa Fluor 647-labeled LEPR (red). Scale bar = 20 μm. (I) Grayscale colocalization analysis of the images shown in (H), (a) PCC (HDPC) = 0.5298 and (b) PCC (MDPC) = 0.5263. All results are presented as means ± SDs (* P < 0.05 and *** P < 0.001). DAPI, 4′,6-diamidino-2-phenylindole; HF, hair follicle; HDPC, human dermal papilla cell; MDPC, mouse dermal papilla cell; mEK, mouse epidermal keratinocyte; FITC, fluorescein isothiocyanate; PCC, Pearson correlation coefficient.

Article Snippet: Intracellular LEPR was labeled with a rabbit anti-LEPR protein monoclonal antibody (1:100, HUABIO), followed by an Alexa Fluor 561 goat anti-rabbit secondary antibody incubation.

Techniques: Expressing, Biomarker Discovery, Binding Assay, Western Blot, Imaging, Incubation, Labeling

Journal: Biomaterials Research

Article Title: Oxidative Stress and Hormone-Regulated Dermal Papilla Cell-Targeted Nanomodulators: Reverse Cellular Senescence for Androgenetic Alopecia Therapy

doi: 10.34133/bmr.0333

Figure Lengend Snippet: Transdermal retention and cellular uptake of L-LPs. (A) CLSM images of pig skin after the application of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil)-labeled LPs or L-LPs for 12 and 24 h. Scale bar = 200 μm. (B) In vitro skin retention of liposomes and free drug ( n = 3). (C) FCM analysis and CLSM images of DPCs exposed to LPs or L-LPs for 2 or 6 h. Scale bar = 50 μm ( n = 3). (D) Colocalization images of LEPR and L-LPs. Scale bar = 100 μm (* P < 0.05 and *** P < 0.001). CLSM, confocal laser scanning microscope; FCM, flow cytometry.

Article Snippet: Intracellular LEPR was labeled with a rabbit anti-LEPR protein monoclonal antibody (1:100, HUABIO), followed by an Alexa Fluor 561 goat anti-rabbit secondary antibody incubation.

Techniques: Labeling, In Vitro, Liposomes, Laser-Scanning Microscopy, Flow Cytometry