Fig 2: 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.

Fig 3: Fer-1 and DFO attenuated osteoarthritis (OA) development through inhibiting chondrocytes ferroptosis. (a) Representative safranin O-fast green images of osteoarthritic knee joints, that were collected 8 weeks after ACLT surgery. Yellow arrowheads indicate articular cartilage degradation. n = 5; Scale bar, left, 200 μm; right, 50 μm. (b) The severity of OA-like phenotype was analyzed using the Osteoarthritis Research Society International (OARSI) score system. n = 5. (c) and (d) Osteophytes were semi-quantified by evaluating the osteophyte formation score consisting of two domains, size (c) and maturity (d). n = 5. (e) Quantitative micro-CT analysis of tibial subchondral trabecular bone with bone volume fraction. n = 5. (f) Quantitative micro-CT analysis of tibial subchondral bone plate thickness. n = 5. (g) Three-dimensional models of mice knee joints. Red arrow shows osteophyte formation. Scale bar, 500μm. (h) Mitochondrial morphology of mice cartilage was observed using transmission electron microscopy. Scale bar, left, 2 μm; right, 500 nm. (i-l) Quantification of mRNA levels for cartilage anabolism related genes (Col2a1, Acan, Sox9, Prg4), catabolism related genes (Mmp3, Mmp13, Adamts1, Adamts5), ferroptosis related genes (Gpx4, Acsl4, Ptgs2, Fth1) and inflammatory related genes (Ccl2, IL-6) in articular cartilage obtained from sham, vehicle, and Fer-1, DFO, Nec-1 treated mice, respectively. 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.

Fig 4: Evaluation of the effect of antagomiR-221 treatment on intervertebral disc cells (IVD) cells. (A) Before transfection the expression levels of miR-221 were measured in P0 cells from MILD and SEVERE IDD by qRT-PCR. Data are presented as fold changes relative to control represented by human freshly isolated chondrocytes (hCH). Results represent mean ± SD (hCH group, n = 6; MILD group, n = 7; SEVERE group, n = 9). Exact P-values are reported. (B) miR-221 levels were measured during de-differentiation process from passage 0 (P0) to passage (P4). Data are presented as fold changes relative to P0 cells. (P0 group, n = 6; P2 group, n = 6; P4 group, n = 6). Exact P-values are reported. Monolayered P2 cells were then transfected with antagomiR-221 (black column), a scrambled oligonucleotide (antagomiR-scr) (gray column) or remained untreated (white column) (C, D, E). (C) The efficiency of miR-221 knockdown was determined by qRT-PCR (C) and data are presented as fold change respect to control untreated cells ±SD (n = 10). Exact P-values are reported. Cell viability of transfected cells was determined by double staining with Calcein-AM/propidium iodide. The green fluorescence indicates the presence of calcein-labeled live cells, while propidium iodide-labeled dead cells are revealed by red fluorescence. Merged photomicrographs are reported. Scale bars: 20 μm. Expression of COL2A1, SOX9, ACAN and TRPS1 chondrogenic markers was assessed by qRT-PCR D) and immunocytochemistry E). (D) The expression of typical chondrogenic markers evaluated at mRNA level. mRNA data are presented as fold change relative to untreated cells. Results represent means ± SD (n = 10). Exact P-values are reported. (E) The expression of typical chondrogenic markers evaluated at protein level. Representative optical photomicrographs of COL2A1, SOX9, ACAN and TRPS1 immunostaining are reported. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software and expressed as means of pixels per one hundred cells ±SD (n = 10). Exact P-values are reported. Scale bars: 20 μm.

Fig 5: Immunofluorescence analysis of Aggrecan and type II collagen expression in NHDFs cultured with alginate beads. Paraffin-embedded, fixed sections were subjected to immunohistochemical analysis using antibodies specific for ACAN and type II collagen: (a) ACAN; (b) COL2. Control indicates empty pcDNA3.1 transfected NHDFs.