Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Oxymatrine Protects Chondrocytes against IL-1 β -triggered Apoptosis in Vitro and Inhibits Osteoarthritis in Mice Model

doi: 10.1155/2022/2745946

Figure Lengend Snippet: Impact of OMT on the IL-1 β -triggered apoptosis in chondrocytes. (a) Western blot results of cleaved-caspase-3, Bcl-2, Bax. (b) Results of flow cytometry. ∗ P < 0.05 and ∗∗ P < 0.01 vs IL-1 β -induced group; # P < 0.05 and ## P < 0.01 vs control group.

Article Snippet: The CCK-8 kit was supplied by Beijing Solarbio Technology Co. Ltd. Antibodies specific for β -actin (Abcam, # ab8227), Bax (Bioss, # bs-0127R), type II collagen (Bioss, # bs-11929R), Bcl-2 (Bioss, # bs-0032R), type X collagen (Bioss, # bs-20085R), cleaved-caspase-3 (Abcam, # ab214430), cleaved-caspase-9 (CST, # 9507S), I κ B α (CST, # 9242S), p-I κ B α (CST, # 2859T), p65 (CST, # 8242T), p-p65 (CST, # 3303T), MMP-13 (Proteintech, # 18165-1-AP), and MMP-9 (Proteintech, # 10375-2-AP) were used in this study.

Techniques: Western Blot, Flow Cytometry

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Oxymatrine Protects Chondrocytes against IL-1 β -triggered Apoptosis in Vitro and Inhibits Osteoarthritis in Mice Model

doi: 10.1155/2022/2745946

Figure Lengend Snippet: TUNEL and IHC staining for BCL-2 and Bax show the effect of OMT on progression of ACLT-triggered OA mice. (a) TUNEL results of mice in each group 8 weeks following surgery. Scale bar: 100 μ m. Right: results of statistical analyses of apoptotic chondrocyte rate. (b) IHC staining for BCL-2 and Bax expression of mice in each group 8 weeks following surgery. Scale bar: 100 μ m. Right: results of statistical analyses displayed as the percentage of Bcl-2 or Bax-positive expression area within the total area. ∗ P < 0.05, and ∗∗∗ P < 0.001 vs vehicle group; # P < 0.05 and ### P < 0.001 vs sham group.

Article Snippet: The CCK-8 kit was supplied by Beijing Solarbio Technology Co. Ltd. Antibodies specific for β -actin (Abcam, # ab8227), Bax (Bioss, # bs-0127R), type II collagen (Bioss, # bs-11929R), Bcl-2 (Bioss, # bs-0032R), type X collagen (Bioss, # bs-20085R), cleaved-caspase-3 (Abcam, # ab214430), cleaved-caspase-9 (CST, # 9507S), I κ B α (CST, # 9242S), p-I κ B α (CST, # 2859T), p65 (CST, # 8242T), p-p65 (CST, # 3303T), MMP-13 (Proteintech, # 18165-1-AP), and MMP-9 (Proteintech, # 10375-2-AP) were used in this study.

Techniques: TUNEL Assay, Immunohistochemistry, Expressing

Journal: Antioxidants

Article Title: Diabetes Upregulates Oxidative Stress and Downregulates Cardiac Protection to Exacerbate Myocardial Ischemia/Reperfusion Injury in Rats

doi: 10.3390/antiox9080679

Figure Lengend Snippet: Western plot ( A ) and immunohistochemistry ( E–F ) of myocardial caspase 3, and PARP and apoptosis TUNEL stain in IR myocardium. The statistic data in Caspase 3 ( B ), PARP ( C ) and TUNEL positive nuclei ( D ) are indicated. Data are expressed as mean ± SEM ( n = 3) using the single values. * p < 0.05 vs. CON. a p < 0.05 vs. STZ. b p < 0.05 vs. IR CON.

Article Snippet: The expression of β-actin (#4970, 1:1000; Cell Signaling Technology, MA, USA), 4-HNE (bs-6313R, 1:1000; Bioss, MA, USA), BAG3 (NBP2-27398, 1:2000; Novus, CO, USA), Bcl-2 (bs-0032R, 1:1000; Bioss, MA, USA), caspase 3 (bs-0081R, 1:1000; Bioss, MA, USA), p-eNOS (ser1177, 1:1000; Cell Signaling Technology, MA, USA) and PARP (#9532, 1:1000; Cell Signaling Technology, MA, USA) from the damaged heart infarct areas after ischemia/reperfusion injury were evaluated by Western blotting as described before [ ].

Techniques: Western Blot, Immunohistochemistry, TUNEL Assay, Staining

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: Mice with Nsd1 knockout in mesenchymal progenitors showed impaired cartilage development. a mRNA levels of H3K36 methyltransferases and chondrocyte differentiation marker genes were determined by qRT-PCR in micromasses at different differentiation time points. The values are presented as the means ± SEMs, n = 4. * P < 0.05, ** P < 0.01, ns means not significant. The inset shows Alcian blue staining results of micromasses cultured for 1, 4, and 7 days with chondroprogenitor cells. Scale bar = 2 mm. b SO staining results of E11.5 limb buds. Scale bar = 100 μm. c Whole-mount in situ hybridization (WISH) results for Col2 in E12.5 embryos (top) and sections of forelimbs (bottom). The dashed purple lines show the digits already present. Scale bar (top) = 500 μm, scale bar (bottom) = 200 μm. d SO staining results of E13.5 (first line), E14.5 (second line), E15.5 (third line), and E16.5 (fourth line) femur sections from WT and Nsd1 f/f ;Prx1-Cre mice. The dashed black lines show the borders between hypertrophic chondrocytes and the primary ossification center. Scale bar = 200 μm. SO staining of P7 ( e ) and P14 ( f ) hindlimb sections from WT and Nsd1 f/f ;Prx1-Cre mice. Scale bar (top) = 200 μm. Scale bar (bottom) = 500 μm. g Whole-mount in situ hybridization (WISH) results for Col2 in E12.5 embryos (top) and sections of forelimbs (bottom). The dashed purple lines show the digits already present. Scale bar (top) = 500 μm, scale bar (bottom) = 200 μm. h SO staining results of E13.5 (top) and E15.5 (bottom) femur sections from WT, Nsd1 f/f ;Col2-Cre mice. The dashed black lines show the borders between hypertrophic chondrocytes and the primary ossification center. Scale bar = 200 μm. SO staining of P7 ( i ) and P14 ( j ) hindlimb sections from WT and Nsd1 f/f ;Col2-Cre mice. Scale bar (top) = 200 μm, Scale bar (bottom) = 500 μm

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: Knock-Out, Marker, Quantitative RT-PCR, Staining, Cell Culture, In Situ Hybridization

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: Nsd1 deficiency in mesenchymal progenitors led to skeletal growth defects in mice. Gross images of 1-month-old WT, Nsd1 f/f ;Prx1-Cre ( a ) and Nsd1 f/f ;Col2-Cre ( b ) mice. Pictures of hindlimbs (left) and quantitative statistics (right) of hindlimb length in 1-month-old WT, Nsd1 f/f ;Prx1-Cre ( c ) and Nsd1 f/f ;Col2-Cre ( d ) mice. Scale bar = 2 mm. The values are presented as the means ± SEMs, n = 6. * P < 0.05, ns means not significant. Safranin O (SO) staining results ( e , g ) and growth plate quantification data ( f , h ) of tibia sections from 1-month-old WT, Nsd1 f/f ;Prx1-Cre ( e , f ) and Nsd1 f/f ;Col2-Cre ( g , h ) mice. Scale bar (top) = 100 μm. Scale bar (bottom) = 50 μm. The values are presented as the means ± SEMs, n = 6. * P < 0.05, ** P < 0.01, ns means not significant

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: Staining

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: Mice with Nsd1 knockout in mesenchymal progenitors showed impaired fracture healing. a Radiographs of fractured femurs from WT and Nsd1 f/f ;Prx1-Cre mice at different days post fracture (dpf). b Quantitative analysis of formed calluses at different days post fracture (dpf). n = 5. Alcian blue/eosin staining ( c ) and quantitative results ( d ) of callus sections. The dashed black lines show the location of the callus. Scale bar = 500 μm. n = 5. Immunofluorescence staining ( e ) and quantitative results ( f ) of type II collagen in callus sections. The dashed white lines show the location of the callus. Scale bar= 50 µm . n = 5. g Micro-CT images of calluses in WT and Nsd1 f/f ;Prx1-Cre mice at 18 dpf. h Quantitative statistics of micro-CT results of calluses. n = 3. i Radiographs of fractured femurs in WT and Nsd1 f/f ;Col2-Cre mice at different days post fracture (dpf). j Quantitative analysis of formed calluses at different days post fracture (dpf). n = 5. Alcian blue/eosin staining ( k ) and quantitative results ( l ) of callus sections. The dashed black lines show the location of the callus. Scale bar = 500 μm. n = 5. m Micro-CT images of calluses in WT and Nsd1 f/f ;Col2-Cre mice at 21 dpf. n Quantitative statistics of micro-CT results of calluses. BV bone volume, BS bone surface, Tb.N trabecular bone number. n = 3. The values are presented as the means ± SEMs. * P < 0.05, ** P < 0.01, ns means not significant

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: Knock-Out, Staining, Immunofluorescence, Micro-CT

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: Chondroprogenitor cells with Nsd1 knockout showed impaired chondrocyte differentiation and increased proliferation. a Gross images of pellets formed by chondroprogenitor cells from neonatal mice. Scale bar = 1 mm. b HE staining (top), SO staining (middle), and Col2 in situ hybridization (bottom) results of sections from pellets formed by chondroprogenitor cells. Scale bar = 20 μm. c Alcian blue staining results of micromasses cultured for 1, 4, and 7 days with chondroprogenitor cells. Scale bar = 2 mm. d Quantitative analysis of Alcian blue staining. The values are presented as the means ± SEMs, n = 4. ** P < 0.05, ns means not significant. qRT-PCR results for Nsd1 ( e ) and chondrocyte differentiation marker genes, including Sox9 ( f ), Col2 ( g ), and Acan ( h ), in micromasses cultured for 1, 4, and 7 days with chondroprogenitor cells. The values are presented as the means ± SEMs, n = 4. * P < 0.05, ** P < 0.01, ns means not significant. i Crystal violet staining results of chondroprogenitor cells cultured for 1, 3, 5, and 7 days. Scale bar = 5 mm. j Quantification of crystal violet staining. The values are presented as the means ± SEMs, n = 6. ** P < 0.01, ns means not significant

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: Knock-Out, Staining, In Situ Hybridization, Cell Culture, Quantitative RT-PCR, Marker

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: Sox9 was regulated by NSD1 through H3K36 methylation. a Heat map of RNA-seq results for Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells. b Pie chart showing the percentages of differentially expressed genes between Egfp and Cre samples. c Normalized reads of H3K36me2 ChIP-seq analyses in Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells from 2 kb upstream of the TSS to 2 kb downstream of the TSS in the genome. d Venn diagram showing the numbers of genes with decreased expression in RNA-seq data (pink), genes with decreased H3K36me2 occupancy in ChIP-seq data (green), and overlapping genes (yellow). e Heat map and annotation of transcription factors from the set of overlapping genes. f Western blot analysis of SOX9 in Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells. g Immunohistochemical assay of SOX9 in growth plate sections from P7 mice. Scale bar (left) = 100 μm, scale bar (right) = 5 μm. h H3K36me2 binding peaks on Sox9 in Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells from the H3K36me2 ChIP-seq assay. i ChIP-PCR assay of H3K36me1 (left) and H3K36me2 (right) occupancy of Sox9 . The values are presented as the means ± SEMs, n = 3. * P < 0.05, ** P < 0.01, ns means not significant. j Alcian blue staining results of micromass culture with chondroprogenitor cells without or with Sox9 overexpression. Scale bar = 2 mm. k qRT-PCR results of Sox9 , Col2 , and Acan in micromass culture. The values are presented as the means ± SEMs, n = 4. * P < 0.05, ** P < 0.01

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: Methylation, RNA Sequencing Assay, Expressing, ChIP-sequencing, Western Blot, Immunohistochemical staining, Binding Assay, Staining, Over Expression, Quantitative RT-PCR

Journal: Bone Research

Article Title: H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

doi: 10.1038/s41413-021-00148-y

Figure Lengend Snippet: NSD1 directly regulated Hif1α . a Heat map of Hif1α and its target genes from the RNA-seq results of Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells. b Western blot analysis of the HIF1α level in Egfp - and Cre -expressing immortalized Nsd1 f/f chondroprogenitor cells. c Immunofluorescence analysis of HIF1α in limb buds of E15.5 mice. Scale bar = 100 μm. d NSD1 binding peaks on Hif1α in ATDC5 cells from the Flag-NSD1 ChIP-seq assay. e ChIP-PCR assay of NSD1 binding on Hif1α . The values are presented as the means ± SEMs, n = 3. ** P < 0.01, ns means not significant. f Luciferase assay of the NSD1-specific binding (NSB) region in the Hif1α promoter in C3H10 cells treated with NSD1. The values are presented as the means ± SEMs, n = 3. * P < 0.05, ** P < 0.01, ns means not significant. g Model that summarizes our findings on the role of NSD1 in regulating Sox9 directly and indirectly. On the one hand, NSD1 can directly promote Sox9 expression by regulating the levels of H3K36me1/2 in the Sox9 promoter region. On the other hand, NSD1 directly binds to the promoter region of Hif1α , activating Hif1α transcription and ultimately promoting Sox9 expression

Article Snippet: Antibodies specific for the following molecules were used: NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), HIF1α (WB: Novus, NB100-134; IF: Bioss, bs-0737R), and Flag (Sigma, F1804).

Techniques: RNA Sequencing Assay, Expressing, Western Blot, Immunofluorescence, Binding Assay, ChIP-sequencing, Luciferase