Fig 2: TNF-a and CHX treatment elevates chondrocyte death and inhibits chondrocyte migration in vitro by initiating fatal mitochondrial fission and interrupting the NA4A1-AMPK signaling pathway. TNF-a, tumor necrosis factor-a; NR4A1, nuclear receptor subfamily 4 group A member 1; AMPK, AMP-activated protein kinase; mPTP, mitochondrial permeability transition pore; CHX, cycloheximide.

Fig 3: NR4A1 contributes to mitochondrial dysfunction via excessive mitochondrial fission. (A) The percentage of double-stranded mtDNA indicates mtDNA strand breaks. (B) mtDNA copy number was assessed by complex IV segment. (C) The transcript level of mtDNA was measured by ND1, the light chain of mtDNA, and COXI, and the heavy chain of mtDNA. (D) Change in ATP contents. (e) The expression of mitochondrial eTCx was measured by western blot analysis. (F) TNF-a and CHX inhibited the content of ETCx. (G) Changes in ETCx I, II and V activities. (H) The effect of NR4A1 on state 3 respiration in chondrocytes under TNF-a and CHX stimulation. (I) The effect of NR4A1 on state 4 respiration in chondrocytes under TNF-a and CHX stimulation. (J) Change of mitoROS (scale bar, 10 µm). *P<0.05 vs. ctrl group. #P<0.05 vs. TNF-a and CHX group. ctrl, control; TNF-a, tumor necrosis factor-a; NR4A1, nuclear receptor subfamily 4 group A member 1; si, small interfering RNA; TNF-a, tumor necrosis factor-a; mtDNA, mitochondrial DNA; mitoROS, mitochondrial reactive oxygen species; CII-core2, complex III subunit core; CII-30, complex II; CIV-II, complex IV subunit II; CI-20, complex I subunit NDUFB8; ND-1, NADH dehydrogenase subunit 1; COX1, cytochrome c oxidase subunit I; eTCx, electron transport chain complexes; CHX, cycloheximide.

Fig 4: NR4A1 regulates mitochondrial fission via the AMPK/Drp1 pathway. AI, an activator of the AMPK pathway, was used as the positive control and cC, an inhibitor of the AMPK pathway, was used as the negative control. (A) Western blot analysis were used to measure the expression of (B) p-AMPK, (C) cyto-Drp1, (D) mito-Drp1 and (e) p-Drp1. (F) NR4A1 represses AMPK activation as indicated by an increased AMP/ATP ratio following transfection with siNR4A1. *P<0.05 vs. ctrl group. #P<0.05 vs. TNF-a and CHX group. &P<0.05 vs. TNF-a and CHX+siNR4A1 group. AI, AICAR; cC, compound C; ctrl, control; TNF-a, tumor necrosis factor-a; si, small interfering RNA; p, phoshphorylated; t, total; cyto, cytoplasmic; mito, mitochondrial; AMPK, AMP-activated protein kinase; Drp1, dynamin-related protein 1; NR4A1, nuclear receptor subfamily 4 group A member 1; CHX, cycloheximide.

Fig 5: NR4A1 upregulation is associated with chondrocyte death in osteoarthritis. (A) Cell viability was measured by MTT assay. Treatment with TNF-a and CHX for 24 h reduced chondrocyte viability in a time-dependent manner. (B) The expression of NR4A1 was measured by western blotting. (C) NR4A1 expression was significantly increased by TNF-a and CHX treatment. (D) Transfection efficiency was confirmed by western blotting. (e) The images of western blotting. (F) Knockdown of NR4A1 had no effect on chondrocyte viability. The role of NR4A1 on chondrocyte survival was measured by (G) lDH release assay, (H) MTT assay and (I) Annexin V/PI flow cytometric analysis. (J) The images of Annexin V/PI flow cytometric analysis. *P<0.05 vs. ctrl group. #P<0.05 vs. TNF-a and CHX group. ctrl, control; TNF-a, tumor necrosis factor-a; NR4A1, nuclear receptor subfamily 4 group A member 1; lDH, lactate dehydrogenase; PI, propidium iodide; si, small interfering RNA; CHX, cycloheximide.