Journal: Nature Communications

Article Title: Dysregulation of the NUDT7-PGAM1 axis is responsible for chondrocyte death during osteoarthritis pathogenesis

doi: 10.1038/s41467-018-05787-0

Figure Lengend Snippet: NUDT7 , a peroxisomal gene, is suppressed during osteoarthritis (OA) pathogenesis. a Staining with Alcian blue or safranin O and immunohistochemistry of Collagen C1-2C, Aggrecan Neoepitope, and matrix metalloproteinase (MMP)-13 using non-OA and OA cartilage from human patients with OA pathogenesis ( n = 5 per group). Scale bar, 50 μm. b Transcription levels of 94 peroxisome-related genes in human OA chondrocytes ( n = 15 per group) compared with those in normal chondrocytes ( n = 8 per group). c Immunostaining of NUDT7, and the mRNA level of NUDT7 in human OA chondrocytes ( n = 20 per group) compared with that in normal chondrocytes ( n = 13 per group). Scale bar, 50 μm. d Transcription level of NUDT7 in normal chondrocytes infected with lentivirus containing each NUDT7 short hairpin RNA (shRNA). e Transcription level of matrix-degrading enzymes in normal chondrocytes infected with lentivirus containing an NUDT7 shRNA ( shNUDT7 ) ( n = 3). f , g Analysis of cell proliferation ( n = 4 per group) and apoptotic cell death ( n = 6 per group) in normal chondrocytes infected with lentivirus containing NUDT7 or NUDT7 shRNA. h , i Activities of catalase and glutathione peroxidase (GPx) in normal chondrocytes infected with lentivirus containing NUDT7 or NUDT7 shRNA ( n = 6 per group). Values are means + s.d. An unpaired Student’s t test was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used in this study; rabbit anti-Collagen C1–2C (IBEX Pharmaceuticals, Cat #50–1035, 1:100), rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, Cat #9664, 1:1000), rabbit anti-cleaved Caspase-9 (Cell Signaling Technology, Cat #9509, 1:1000), rabbit anti-PMP70 (Abcam, Cat #ab3421, 1:500), rabbit anti-FASN (Abcam, Cat #ab22759, 1:200), rabbit anti-H3K4me3 (Abcam, Cat #ab8580, 1:250), mouse anti-H3K27me3 (Abcam, Cat #ab6002, 1:250), rabbit anti-MMP13 (Biovision, Cat #3533, 1:200), rabbit anti-Aggrecan Neoepitope (NOVUS, Cat #100–74350), anti-human NUDT7 (Mybiosource, Cat #MBS416528), rabbit anti-PGAM1 (LSbio, Cat #LS-C143463, 1:200), rabbit anti-vinculin (Invitrogen, Cat #PA5-29688, 1:1000), customized rabbit anti-mouse NUDT7 (Younginfrontier, Korea), HRP-conjugated goat anti-rabbit IgG (Cat #ADI-SAB-300-J, Enzo Lifesciences), and goat anti-mouse IgG polyclonal antibodies (Cat # #ADI-SAB-100-J, Enzo Lifesciences).

Techniques: Staining, Immunohistochemistry, Immunostaining, Infection, shRNA

Journal: Nature Communications

Article Title: Dysregulation of the NUDT7-PGAM1 axis is responsible for chondrocyte death during osteoarthritis pathogenesis

doi: 10.1038/s41467-018-05787-0

Figure Lengend Snippet: Nudt7 −/− mice have severe cartilage degradation and chondrocyte degeneration. a Staining of Collagen C1-2C, Aggrecan Neoepitope, and matrix metalloproteinase (MMP)-13 of paraffin sections from the articular cartilage of Nudt7 +/+ and Nudt7 −/− mice ( n = 5 per group). Scale bars, 50 μm. b Analysis of cartilage thickness, Mankin score, and chondrocyte number using destabilization of the medial meniscus (DMM)-induced Nudt7 +/+ or Nudt7 −/− mice under a normal- (NCD) or phytol-enriched diet (PED) from Fig. 2a ( n = 5 per group). c Positive cell number of Collagen C1-2C, Aggrecan (ACAN) Neoepitope, and MMP-13 in DMM-induced Nudt7 +/+ or Nudt7 −/− mice under a NCD or PED from Fig. 2a ( n = 5 per group). d Staining of Alcian blue, PMP70, BODIPY 493/508 using iMACs of Nudt7 +/+ and Nudt7 −/− mice at postnatal day 6 under a control (CON) or 50 mM phytol treatment (phytol; n = 5 per group). Scale bars, 10 μm. e Quantification of Alcian blue staining extracted with 6M Guanidine-HCl was measured in 650 nm absorbance ( n = 5 per group). f Analysis of apoptotic cell death in iMACs of Nudt7 −/− mice at postnatal day 6 under CON or phytol conditions compared with those of Nudt7 +/+ mice ( n = 6 per group). g Transcription level of MMP-13 and -9, ADAMTS-4 and -5 in CON and phytol in iMACs of Nudt7 −/− mice at postnatal day 6 under CON or phytol conditions compared with those of Nudt7 +/+ mice ( n = 3 per group). Values are means + s.d. A one-way ANOVA was used for statistical analysis ( b , c , e and f ). Values are means + s.d. An unpaired Student’s t test was used for statistical analysis ( g ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used in this study; rabbit anti-Collagen C1–2C (IBEX Pharmaceuticals, Cat #50–1035, 1:100), rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, Cat #9664, 1:1000), rabbit anti-cleaved Caspase-9 (Cell Signaling Technology, Cat #9509, 1:1000), rabbit anti-PMP70 (Abcam, Cat #ab3421, 1:500), rabbit anti-FASN (Abcam, Cat #ab22759, 1:200), rabbit anti-H3K4me3 (Abcam, Cat #ab8580, 1:250), mouse anti-H3K27me3 (Abcam, Cat #ab6002, 1:250), rabbit anti-MMP13 (Biovision, Cat #3533, 1:200), rabbit anti-Aggrecan Neoepitope (NOVUS, Cat #100–74350), anti-human NUDT7 (Mybiosource, Cat #MBS416528), rabbit anti-PGAM1 (LSbio, Cat #LS-C143463, 1:200), rabbit anti-vinculin (Invitrogen, Cat #PA5-29688, 1:1000), customized rabbit anti-mouse NUDT7 (Younginfrontier, Korea), HRP-conjugated goat anti-rabbit IgG (Cat #ADI-SAB-300-J, Enzo Lifesciences), and goat anti-mouse IgG polyclonal antibodies (Cat # #ADI-SAB-100-J, Enzo Lifesciences).

Techniques: Staining

Journal: Nature Communications

Article Title: Dysregulation of the NUDT7-PGAM1 axis is responsible for chondrocyte death during osteoarthritis pathogenesis

doi: 10.1038/s41467-018-05787-0

Figure Lengend Snippet: NUDT7 deficiency affects glycolysis via upregulation of phosphoglycerate mutase 1 (PGAM1) expression during osteoarthritis (OA) pathogenesis. a Comparison of RNA sequencing between human OA chondrocyte and cartilage of Nudt7 −/− mice, and possible pathological signaling pathways using Ingenuity Pathway Analysis (IPA). b Transcription level (ΔCt value) of PGAM1 in human OA chondrocytes ( n = 15) compared with that in normal chondrocytes ( n = 8). c Immunohistochemistry of PGAM1 in human OA cartilages ( n = 5 per group) and cartilages of Nudt7 −/− mice ( n = 5 per group) under NCD or PED conditions. Scale bars, 50 μm. d Positive cell number of PGAM1 in human OA cartilages ( n = 5 per group) and cartilages of Nudt7 −/− mice ( n = 5 per group) under NCD or PED conditions from Fig. 3c. e – g Analysis of 2-phosphoglycerate, acetyl-CoA, and malonyl-CoA in immature murine articular chondrocytes (iMACs) of Nudt7 −/− mice at postnatal day 6 ( n = 6 per group). Values are means + s.d. An unpaired Student’s t test was used for statistical analysis. ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used in this study; rabbit anti-Collagen C1–2C (IBEX Pharmaceuticals, Cat #50–1035, 1:100), rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, Cat #9664, 1:1000), rabbit anti-cleaved Caspase-9 (Cell Signaling Technology, Cat #9509, 1:1000), rabbit anti-PMP70 (Abcam, Cat #ab3421, 1:500), rabbit anti-FASN (Abcam, Cat #ab22759, 1:200), rabbit anti-H3K4me3 (Abcam, Cat #ab8580, 1:250), mouse anti-H3K27me3 (Abcam, Cat #ab6002, 1:250), rabbit anti-MMP13 (Biovision, Cat #3533, 1:200), rabbit anti-Aggrecan Neoepitope (NOVUS, Cat #100–74350), anti-human NUDT7 (Mybiosource, Cat #MBS416528), rabbit anti-PGAM1 (LSbio, Cat #LS-C143463, 1:200), rabbit anti-vinculin (Invitrogen, Cat #PA5-29688, 1:1000), customized rabbit anti-mouse NUDT7 (Younginfrontier, Korea), HRP-conjugated goat anti-rabbit IgG (Cat #ADI-SAB-300-J, Enzo Lifesciences), and goat anti-mouse IgG polyclonal antibodies (Cat # #ADI-SAB-100-J, Enzo Lifesciences).

Techniques: Expressing, RNA Sequencing Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: Dysregulation of the NUDT7-PGAM1 axis is responsible for chondrocyte death during osteoarthritis pathogenesis

doi: 10.1038/s41467-018-05787-0

Figure Lengend Snippet: Upregulation of phosphoglycerate mutase 1 ( PGAM1 ) expression induces pro-inflammatory cytokines. a Immunostaining of interleukin (IL)-1β in human OA chondrocyte compared with non-OA chondrocyte ( n = 5 per group). b Immunostaining of IL-1β cartilage of DMM-induced Nudt7 −/− mice ( n = 5 per group). c Transcription levels of IL-1β, -6 , and TNFα in iMACs of Nudt7 −/− mice at postnatal day 6 under CON and phytol conditions ( n = 3 per group). d Immunoblotting of cleaved caspase-3 and -9 with the introduction of PGAM1 into iMACs of Nudt7 +/+ mice. e , f Analysis of apoptotic cell death and cell proliferation with the introduction of PGAM1 into iMACs of Nudt7 +/+ mice ( n = 6 per group). g Transcriptional level of IL-1β , -6, and TNFα in iMACs of Nudt7 +/+ mice at postnatal day 6 transfected with PGAM1 dose dependent ( n = 3 per group). h Transcriptional levels of MMP-13, COL2A1, ACAN , and COMP transfected with PGAM1 . Scale bars, 50 μm. Values are means + s.d. An unpaired Student’s t test was used for statistical analysis. ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used in this study; rabbit anti-Collagen C1–2C (IBEX Pharmaceuticals, Cat #50–1035, 1:100), rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, Cat #9664, 1:1000), rabbit anti-cleaved Caspase-9 (Cell Signaling Technology, Cat #9509, 1:1000), rabbit anti-PMP70 (Abcam, Cat #ab3421, 1:500), rabbit anti-FASN (Abcam, Cat #ab22759, 1:200), rabbit anti-H3K4me3 (Abcam, Cat #ab8580, 1:250), mouse anti-H3K27me3 (Abcam, Cat #ab6002, 1:250), rabbit anti-MMP13 (Biovision, Cat #3533, 1:200), rabbit anti-Aggrecan Neoepitope (NOVUS, Cat #100–74350), anti-human NUDT7 (Mybiosource, Cat #MBS416528), rabbit anti-PGAM1 (LSbio, Cat #LS-C143463, 1:200), rabbit anti-vinculin (Invitrogen, Cat #PA5-29688, 1:1000), customized rabbit anti-mouse NUDT7 (Younginfrontier, Korea), HRP-conjugated goat anti-rabbit IgG (Cat #ADI-SAB-300-J, Enzo Lifesciences), and goat anti-mouse IgG polyclonal antibodies (Cat # #ADI-SAB-100-J, Enzo Lifesciences).

Techniques: Expressing, Immunostaining, Western Blot, Transfection

Journal: Nature Communications

Article Title: Dysregulation of the NUDT7-PGAM1 axis is responsible for chondrocyte death during osteoarthritis pathogenesis

doi: 10.1038/s41467-018-05787-0

Figure Lengend Snippet: Histone 3 lysine trimethylation (H3K4me3) is involved in the transcriptional activation of phosphoglycerate mutase 1 ( PGAM1 ). a , b Immunohistochemistry of H3K4me3 in human OA cartilage ( n = 5 per group; scale bars, 100 μm) and cartilage of Nudt7 −/− mice ( n = 5 per group; scale bars, 200 μm). c Immunocytochemistry of H3K4me3 and counting of H3K4me3-positive cells in immature murine articular chondrocytes (iMACs) of Nudt7 −/− mice ( n = 6 per group; scale bars, 100 μm). d H3K4me3 binding assay on the PGAM1 promoter using a chromatin immunoprecipitation (ChIP) assay ( n = 3 per group). Values are means + s.d. An unpaired Student’s t test was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used in this study; rabbit anti-Collagen C1–2C (IBEX Pharmaceuticals, Cat #50–1035, 1:100), rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, Cat #9664, 1:1000), rabbit anti-cleaved Caspase-9 (Cell Signaling Technology, Cat #9509, 1:1000), rabbit anti-PMP70 (Abcam, Cat #ab3421, 1:500), rabbit anti-FASN (Abcam, Cat #ab22759, 1:200), rabbit anti-H3K4me3 (Abcam, Cat #ab8580, 1:250), mouse anti-H3K27me3 (Abcam, Cat #ab6002, 1:250), rabbit anti-MMP13 (Biovision, Cat #3533, 1:200), rabbit anti-Aggrecan Neoepitope (NOVUS, Cat #100–74350), anti-human NUDT7 (Mybiosource, Cat #MBS416528), rabbit anti-PGAM1 (LSbio, Cat #LS-C143463, 1:200), rabbit anti-vinculin (Invitrogen, Cat #PA5-29688, 1:1000), customized rabbit anti-mouse NUDT7 (Younginfrontier, Korea), HRP-conjugated goat anti-rabbit IgG (Cat #ADI-SAB-300-J, Enzo Lifesciences), and goat anti-mouse IgG polyclonal antibodies (Cat # #ADI-SAB-100-J, Enzo Lifesciences).

Techniques: Activation Assay, Immunohistochemistry, Immunocytochemistry, Binding Assay, Chromatin Immunoprecipitation

Journal: Nature Communications

Article Title: Dysregulation of the NUDT7-PGAM1 axis is responsible for chondrocyte death during osteoarthritis pathogenesis

doi: 10.1038/s41467-018-05787-0

Figure Lengend Snippet: Cartilage destruction by phosphoglycerate mutase 1 ( PGAM1 ) is reversed by co-introduction of NUDT7 . a Staining with safranin O and BODIPY 493/508 using the cartilage of Nudt7 −/− mice infected with lentivirus containing either PGAM1 shRNA ( shPGAM ) or NUDT7 into the joint cavity 10 weeks after destabilization of the medial meniscus (DMM) surgery ( n = 5, Scale bar, 50 μm). b , c Analysis of cartilage thickness, BODIPY 493/508 -positive cells from staining with safranin O, and BODIPY 493/508 from Fig. 6a. d Staining of BODIPY 493/508 , IL-1β, and PGAM1 with the introduction of shPGAM1 or NUDT7 and/or PGAM1 into iMACs of Nudt7 +/+ mice ( n = 6 per group). Scale bars, 10 μm. e – g Analysis of 2-phosphoglycerate, malonyl-CoA, and apoptotic cell death in immature murine articular chondrocytes (iMACs) of Nudt7 −/− mice infected with lentivirus containing shPGAM or NUDT7 and/or PGAM1 ( n = 6 per group). h BODIPY 493/508 staining of human osteoarthritis (OA) chondrocyte infected with lentivirus containing shPGAM or NUDT7 and/or PGAM1 ( n = 6 per group). Scale bars, 10 μm. Values are means + s.d. An unpaired Student’s t test was used for statistical analysis. ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used in this study; rabbit anti-Collagen C1–2C (IBEX Pharmaceuticals, Cat #50–1035, 1:100), rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, Cat #9664, 1:1000), rabbit anti-cleaved Caspase-9 (Cell Signaling Technology, Cat #9509, 1:1000), rabbit anti-PMP70 (Abcam, Cat #ab3421, 1:500), rabbit anti-FASN (Abcam, Cat #ab22759, 1:200), rabbit anti-H3K4me3 (Abcam, Cat #ab8580, 1:250), mouse anti-H3K27me3 (Abcam, Cat #ab6002, 1:250), rabbit anti-MMP13 (Biovision, Cat #3533, 1:200), rabbit anti-Aggrecan Neoepitope (NOVUS, Cat #100–74350), anti-human NUDT7 (Mybiosource, Cat #MBS416528), rabbit anti-PGAM1 (LSbio, Cat #LS-C143463, 1:200), rabbit anti-vinculin (Invitrogen, Cat #PA5-29688, 1:1000), customized rabbit anti-mouse NUDT7 (Younginfrontier, Korea), HRP-conjugated goat anti-rabbit IgG (Cat #ADI-SAB-300-J, Enzo Lifesciences), and goat anti-mouse IgG polyclonal antibodies (Cat # #ADI-SAB-100-J, Enzo Lifesciences).

Techniques: Staining, Infection, shRNA