Fig 1: Detection of intracellular and mitochondrial levels of reactive oxygen species (ROS) in SH-SY5Y cells. (A) MitoSOX/MitoTracker/Hoechst staining images and fluorescence microscopy were used to determine the level of mitochondrial ROS in rotenone-treated SH-SY5Y cells. Hoechst (blue), Mito-Sox (red), and MitoTracker (green). The ROS level was significantly increased in cells not overexpressing GDF15 compared with that in normal control and GDF15-overexpressing cells. Magnification: 400×. (B) Quantification of mitochondrial ROS levels using flow cytometry and Mito-Sox-Red fluorescent probes. (C,D) Intracellular ROS generation (%) in SH-SY5Y cells was assessed with the ROS-sensitive fluorometric probe DCFH-DA using flow cytometry. Data are presented as the mean ± S.E. from three independent experiments, and differences were analyzed with an unpaired student t-test. **P < 0.01, compared to the control group; ##P < 0.01, as compared to rotenone-treated only group.
Fig 2: Expression of GDF15 prevents rotenone-induced toxicity with respect to the mitochondrial membrane potential (MMP) and might require Akt/mTOR-dependent phosphorylation. (A) MMP in SH-SY5Y cells following treatment with rotenone. The MMP, measured using a JC-10 assay, was significantly decreased by rotenone. (B,C) The oxygen consumption rate (OCR) of SH-SY5Y cells was measured using a Seahorse XF24 Extracellular Flux Analyzer. Basal respiration, maximal respiration, and ATP production in SH-SY5Y cells treated with rotenone were significantly lower than those in control and GDF15-overexpressing cells. Also, when compared to rotenone group, it could be found that basal respiration, maximal respiration, and ATP production improve in rotenone treated GDF15 overexpression group. (D,E) The levels of phosphorylation of both Akt and mTOR in SH-SY5Y cells after 3 h treated with various concentrations of the PI3K/Akt specific inhibitor LY294002 (0–10 µM) were assessed by western blotting analysis. (F–H) Western blotting showing increased GDF15 expression after transfection with the GDF15-overexpression plasmid, which increased the phosphorylation of both Akt and mTOR. Inhibition of the PI3K/AKT pathway using LY294002 upregulated the expression levels of p53 and eliminated the protective effect of GDF15. Anti-ß-actin was used as an internal control. Data are presented as the mean ± S.E. (standard error) from three independent experiments, and differences were analyzed with an unpaired student t-test. **P < 0.01, compared to the control group; #P < 0.05, ##P < 0.01, as compared to rotenone-treated only group or rotenone-treated group after transfection with the GDF15 overexpression plasmid.
Fig 3: GDF15 interacts with ErbB2 in chondrocytes. (A) Western blot protein analysis of ErbB2, pErbB2, MAPK14, and pMAPK14 in C20A4 cells treated with or without recombinant human GDF15 (rhGDF15; 10 ng/mL) for 24 h and shERBB2. (B) GDF15 was immunoprecipitated from the cell lysates of IL-1ß-induced C20A4, and the immunoprecipitation product was immunoblotted with an anti-p-ErbB2 antibody. *** p < 0.001.
Fig 4: GDF15 is highly expressed in the tissue and synovial fluid of patients with OA. (A–C) Comparative study of the expression of GDF15 in healthy individuals and OA patients. (D–F) Correlation analysis between GDF15 and MAPK14, p16 as senescence marker, CD31 as angiogenesis marker, and the severity score of OA. (A) Representative image of the staining of GDF15 in the synovial membrane. Scale bar: 100 µm (B) H score quantification through GDF15 immunohistochemistry staining in the synovial membrane of healthy individuals and patients with OA. (C) ELISA for GDF15 detection in the synovial fluid of healthy individuals and patients with OA. (D) Immunohistochemistry staining comparing GDF15, pMAPK14, p16, and CD31 in the synovial membrane of healthy individuals and patients with OA. Scale bar: 100 µm (E) Moderate correlation of GDF15 expression with pMAPK14 (R2 = 0.494), p16 (R2 = 0.584), and CD31 (R2 = 0.561) in the synovial membrane tissues of healthy individuals and patients with OA. (F) Moderate correlation of GDF15 expression in the synovial fluid and patients’ OARSI scores (R2 = 0.494). ** p < 0.01 and *** p < 0.001.
Fig 5: GDF15-nAb treatment attenuates chondrocyte senescence and SASP. (A) GDF15-nAb (1 µM/24 h) inhibited the development of the SASP markers IL-6, IL-8, MMP-13, and Cdkn1a, as observed in the qRT-PCR assay. (B) SA-ß-GAL staining in C20A4 decreased after treatment with GDF15-nAb. Scale bar: 10 µm (C) Western blot assay demonstrating the effect of GDF15-nAb on chondrocytes treated with GDF15-nAb. *** p < 0.001.
Supplier Page from Abcam for Human GDF-15 ELISA Kit