il 3  (Miltenyi Biotec)

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene attenuates osteoarthritis progression through p53-dependent autophagy activation: evidence from network analysis and experimental validation

doi: 10.3389/fphar.2026.1686555

Figure Lengend Snippet: Effects of PT on chondrocyte viability and ECM metabolism under IL-1β stimulation. (A,B) Cell viability determined by CCK-8 assay in chondrocytes treated with increasing PT concentrations (0, 5, 10, 20, 40, 100 μM) for 24 and 48 h without (A) or with (B) IL-1β (10 ng/mL) stimulation; (C) Representative WB images showing ACAN, COL2A1, MMP3 and MMP13 expression; (D–G) Quantitative analysis of protein levels normalized to GAPDH for ACAN (D) , COL2A1 (E) , MMP13 (F) , and MMP3 (G) . Data = mean ± SEM (n = 3). *P < 0.05, ***P < 0.001 vs. control; #P < 0.05, ###P < 0.001 vs. IL-1β.

Article Snippet: In cell viability assays, cells were exposed to PT at concentrations of 0, 5, 10, 20, 40, and 100 μM, with or without interleukin-1β (IL-1β, 10 ng/mL, HY-P7028, MedChemExpress, Shanghai, China), for 24 and 48 h. To assess PT’s effects on ECM metabolism, cells were treated with IL-1β (10 ng/mL) alone or in combination with PT (10, 20, or 40 μM) for 24 h. To investigate the role of autophagy in PT-mediated chondroprotection, cells were divided into five groups: control (vehicle), IL-1β (10 ng/mL), IL-1β + PT (20 μM), IL-1β + rapamycin (RAPA, 100 nM; HY-10219, MedChemExpress, Shanghai, China), and IL-1β + PT + 3 MA (3-methyladenine, 5 mM; HY-19312, MedChemExpress, Shanghai, China).

Techniques: CCK-8 Assay, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene attenuates osteoarthritis progression through p53-dependent autophagy activation: evidence from network analysis and experimental validation

doi: 10.3389/fphar.2026.1686555

Figure Lengend Snippet: PT protects chondrocytes against IL-1β-induced damage via autophagy activation. (A) Representative WB images showing ACAN, COL2A1, MMP3, MMP13, Beclin1, LC3B, and p62 expression; (B–H) Quantitative analysis of protein levels normalized to GAPDH for ACAN (B) , COL2A1 (C) , MMP13 (D) , MMP3 (E) , Beclin1 (F) , LC3II/I ratio (G) , and p62 (H) ; (I) Representative TUNEL staining showing apoptotic cells (red) and nuclei (blue) (scale bar = 50 μm); (J) Percentage of TUNEL-positive cells. Data = mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 vs. control; ###P < 0.001 vs. IL-1β; †P < 0.05, †††P < 0.001 vs. IL-1β + PT.

Article Snippet: In cell viability assays, cells were exposed to PT at concentrations of 0, 5, 10, 20, 40, and 100 μM, with or without interleukin-1β (IL-1β, 10 ng/mL, HY-P7028, MedChemExpress, Shanghai, China), for 24 and 48 h. To assess PT’s effects on ECM metabolism, cells were treated with IL-1β (10 ng/mL) alone or in combination with PT (10, 20, or 40 μM) for 24 h. To investigate the role of autophagy in PT-mediated chondroprotection, cells were divided into five groups: control (vehicle), IL-1β (10 ng/mL), IL-1β + PT (20 μM), IL-1β + rapamycin (RAPA, 100 nM; HY-10219, MedChemExpress, Shanghai, China), and IL-1β + PT + 3 MA (3-methyladenine, 5 mM; HY-19312, MedChemExpress, Shanghai, China).

Techniques: Activation Assay, Expressing, TUNEL Assay, Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene attenuates osteoarthritis progression through p53-dependent autophagy activation: evidence from network analysis and experimental validation

doi: 10.3389/fphar.2026.1686555

Figure Lengend Snippet: PT modulates p53 localization, AMPK/mTOR signaling, and autophagy-related markers in chondrocytes. (A) CETSA results assessing the thermal stability of p53 in C28/I2 cells treated with PT or vehicle across a temperature range of 38 °C–70 °C. Representative WB bands are shown above the normalized thermal aggregation curves; (B) Representative immunofluorescence confocal microscopy images illustrating p53 subcellular localization (red) and DAPI-stained nuclei (blue) in chondrocytes across different treatment groups (scale bar = 10 μm); (C) Representative WB bands showing p53 distribution (nuclear, cytoplasmic, and total), the phosphorylation status of AMPK and mTOR, and autophagy markers (LC3 and p62); (D–K) Quantitative analyses of protein expression levels: total p53/GAPDH (D) , nuclear p53/PCNA (E) , cytoplasmic p53/GAPDH (F) , nuclear/cytoplasmic p53 ratio (G) , p-AMPK/AMPK (H) , p-mTOR/mTOR (I) , p62/GAPDH (J) , and the LC3II/I ratio (K) ; (L) Representative TUNEL staining images showing apoptotic cells (red) and nuclei (blue) (scale bar = 50 μm); (M) Quantitative analysis of the percentage of TUNEL-positive cells across groups. Data = mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 vs. control; ###P < 0.001 vs. IL-1β; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. IL-1β + PT.

Article Snippet: In cell viability assays, cells were exposed to PT at concentrations of 0, 5, 10, 20, 40, and 100 μM, with or without interleukin-1β (IL-1β, 10 ng/mL, HY-P7028, MedChemExpress, Shanghai, China), for 24 and 48 h. To assess PT’s effects on ECM metabolism, cells were treated with IL-1β (10 ng/mL) alone or in combination with PT (10, 20, or 40 μM) for 24 h. To investigate the role of autophagy in PT-mediated chondroprotection, cells were divided into five groups: control (vehicle), IL-1β (10 ng/mL), IL-1β + PT (20 μM), IL-1β + rapamycin (RAPA, 100 nM; HY-10219, MedChemExpress, Shanghai, China), and IL-1β + PT + 3 MA (3-methyladenine, 5 mM; HY-19312, MedChemExpress, Shanghai, China).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Phospho-proteomics, Expressing, TUNEL Assay, Control