Journal: EBioMedicine

Article Title: Contribution of ferroptosis and GPX4’s dual functions to osteoarthritis progression

doi: 10.1016/j.ebiom.2022.103847

Figure Lengend Snippet: GPX4 regulate ECM disassembly genes through MAPK/NFκB pathway. (a) A volcano plot illustrating differentially regulated gene expression from RNA-seq analysis between NC and shGpx4 MCCs. Genes upregulated and downregulated are shown in red and green, respectively. (b) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of downregulated, upregulated, and total genes in shGpx4 transcriptome. (Top 10) (c) GSEA plots evaluating the changes of extracellular matrix related genes. (d) GSEA plots evaluating the changes of extracellular matrix disassembly related genes and heatmap of representative genes. (e) GSEA plots evaluating the changes of extracellular matrix assembly related genes and heatmap of representative genes. (f) Alcian blue staining of ATDC5-shNC and shGpx4 cells after incubation in chondrogenesis media with or without IL-1β (10ng/ml). Left, representative image of staining at day 8 is shown. Scale bar, 0.25 mm. Right, quantification of staining by colorimetry. (g) Protein expression of Adamts5, MMP3, MMP13, COL2A1, ACAN and ACSL4,SLC7A11, SLC3A2 in chondrocytes infected with shNC or shGpx4 analyzed by Western blot. (h) GSEA plots evaluating the changes of MAPK pathway (up) and Erk pathway (down). (i) Protein expression of FAK, MAPK, pMAPK, and NFκB analyzed by Western blot. Data are expressed as mean ± SD, * P< 0.05; ** P< 0.01; *** P< 0.001. Student's t-test and one-way ANOVA were used for comparison between two groups and multiple groups, respectively.

Article Snippet: Proteins were analyzed with antibodies recognizing GPX4 (1:1000, Abcam Cat# ab125066, RRID:AB_10973901), MMP3 (1:1000, Santa Cruz Cat# sc-21732, RRID:AB_627958), MMP13 (1:1000, Santa Cruz Cat# sc-515284), ACAN (1:1000, Santa Cruz Cat# sc-33695, RRID:AB_626650), ACSL4 (1:1000, Santa Cruz Cat# sc-365230, RRID:AB_10843105), COL2A1 (1:1000, Abcam Cat# ab188570), Adamts5(1:1000, Affinity Cat# DF13268, RRID:AB_2846287), SLC3A2 (1:1000, Affinity Cat# DF7468, RRID:AB_2839405), SLC7A11(1:1000, Affinity Cat# DF12509, RRID:AB_2845314), FAK (1:1000, Cell Signaling Technology Cat# 3285, RRID:AB_2269034), MAPK 42/44 (1:1000, Cell Signaling Technology Cat# 4695, RRID:AB_390779), Phospho-MAPK 42/44 (1:1000, Cell Signaling Technology Cat# 4370, RRID:AB_2315112), NFκB (1:1000, Cell Signaling Technology Cat# 6956, RRID:AB_10828935), AKT (1:1000, Cell Signaling Technology Cat# 4685, RRID:AB_2225340), Phospho-AKT (1:1000, Cell Signaling Technology Cat# 4060, RRID:AB_2315049), PI3K (1:1000, Cell Signaling Technology Cat# 4249, RRID:AB_2165248), p38 MAPK (1:1000, Cell Signaling Technology Cat# 8690, RRID:AB_10999090), Phospho-p38 MAPK (1:1000, Cell Signaling Technology Cat# 4511, RRID:AB_2139682) and β-ACTIN (Servicebio Cat# GB11001, RRID:AB_2801259) (Supplementary Table 5).

Techniques: Gene Expression, RNA Sequencing, Staining, Incubation, Colorimetric Assay, Expressing, Infection, Western Blot, Comparison

Journal: EBioMedicine

Article Title: Contribution of ferroptosis and GPX4’s dual functions to osteoarthritis progression

doi: 10.1016/j.ebiom.2022.103847

Figure Lengend Snippet: GPX4 downregulation accelerated OA progression. (a) Representative safranin O-fast green images of mice knee joints. One week before surgery, C57/BL6J mice (8 weeks old) were injected intra-articularly with AAV carrying GPX4-specific shRNA and analyzed 8 weeks after surgery. Scale bar, left, 500 μm; right, 50 μm. (b) Three-dimensional models of mice knee joints. Red arrow shows osteophyte formation. Scale bar, 500μm. (c) The severity of OA-like phenotype was analyzed using the Osteoarthritis Research Society International (OARSI) score system. n = 5. (d) and (e) Osteophytes were semi-quantified by evaluating the osteophyte formation score consisting of two domains, size (D) and maturity (E). n = 5. (f) Quantitative micro-CT analysis of tibial subchondral trabecular bone with bone volume fraction. n = 5. (g) Quantitative micro-CT analysis of tibial subchondral bone plate thickness. n = 5. (h) In OA cartilage, function of system Xc − was inhibited, result in content of GSH and GPX4 expression decreased. Downregulation of GPX4 not only increased the sensitivity of chondrocytes to oxidative stress, but also aggravated extracellular matrix (ECM) degradation potentially through MAPK/NFκB pathway. Data are expressed as mean ± SD, * P< 0.05; ** P< 0.01; *** P< 0.001. Student's t-test and one-way ANOVA were used for comparison between two groups and multiple groups, respectively.

Article Snippet: Proteins were analyzed with antibodies recognizing GPX4 (1:1000, Abcam Cat# ab125066, RRID:AB_10973901), MMP3 (1:1000, Santa Cruz Cat# sc-21732, RRID:AB_627958), MMP13 (1:1000, Santa Cruz Cat# sc-515284), ACAN (1:1000, Santa Cruz Cat# sc-33695, RRID:AB_626650), ACSL4 (1:1000, Santa Cruz Cat# sc-365230, RRID:AB_10843105), COL2A1 (1:1000, Abcam Cat# ab188570), Adamts5(1:1000, Affinity Cat# DF13268, RRID:AB_2846287), SLC3A2 (1:1000, Affinity Cat# DF7468, RRID:AB_2839405), SLC7A11(1:1000, Affinity Cat# DF12509, RRID:AB_2845314), FAK (1:1000, Cell Signaling Technology Cat# 3285, RRID:AB_2269034), MAPK 42/44 (1:1000, Cell Signaling Technology Cat# 4695, RRID:AB_390779), Phospho-MAPK 42/44 (1:1000, Cell Signaling Technology Cat# 4370, RRID:AB_2315112), NFκB (1:1000, Cell Signaling Technology Cat# 6956, RRID:AB_10828935), AKT (1:1000, Cell Signaling Technology Cat# 4685, RRID:AB_2225340), Phospho-AKT (1:1000, Cell Signaling Technology Cat# 4060, RRID:AB_2315049), PI3K (1:1000, Cell Signaling Technology Cat# 4249, RRID:AB_2165248), p38 MAPK (1:1000, Cell Signaling Technology Cat# 8690, RRID:AB_10999090), Phospho-p38 MAPK (1:1000, Cell Signaling Technology Cat# 4511, RRID:AB_2139682) and β-ACTIN (Servicebio Cat# GB11001, RRID:AB_2801259) (Supplementary Table 5).

Techniques: Injection, shRNA, Micro-CT, Expressing, Comparison