Journal: Development (Cambridge, England)

Article Title: Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

doi: 10.1242/dev.018044

Figure Lengend Snippet: (A,B) Forelimbs of PTHrP−/− or wild-type E14.5 embryos cultured for 36 hours in the presence or absence of recombinant Shh protein or cyclopamine. Treated limbs were compared with untreated contralateral control ones. Serial sections of the proximal humerus of the cultured limbs were hybridized with 35S riboprobes of Ihh (A) or Col10a1 (B). The distances from the articular end to the Ihh/Col10a1 domain in the untreated wild-type and PTHrP−/− embryos are indicated by orange lines. This distance was increased in the Shh-treated wild-type humerus but reduced in the Shh-treated PTHrP−/− humerus (white lines) compared with the untreated contralateral controls. This distance was reduced in the cyclopamine-treated wild type, but increased in the PTHrP−/− humerus (green lines) compared with the untreated controlateral controls. (C) Statistical analysis (paired Student′s t-test) of the distance from articular chondrocytes to the hypertrophic zone between treated and untreated contralateral limbs. Numbers (n) of analyzed limbs are indicated. (D) Serial sections of E14.5 distal humeri of the indicated genotypes stained with Safranin O (upper panel) and hybridized with a 35S Col10a1 riboprobe (lower panel) to detect chondrocyte hypertrophy. The distance from the articular end to the Col10a1 expression domain (orange lines) in the PTHrP−/− embryos was reduced compared with that in the wild-type embryos. This distance in the PTHrP−/−;UAS-cIhh;Col2a1-Gal4 double mutant was further reduced compared with that of the PTHrP−/− embryos.

Article Snippet: Primary antibodies (anti-phospho-Smad1 goat polyclonal IgG (Santa Cruz) at 1:100 and anti-Col2a1 goat polyclonal IgG (Santa Cruz) at 1:200 were used for immunohistochemistry, and signals were detected using the ABC kits (Vector Laboratories) and DAB (Sigma).

Techniques: Cell Culture, Recombinant, Staining, Expressing, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

doi: 10.1242/dev.018044

Figure Lengend Snippet: (A) Skeletal preparation of embryos at E14.5. Alizarin Red stains mineralized cartilage and bone tissues; Alcian Blue stains unmineralized cartilage. A higher magnification view of the forelimb is shown in the lower panel. S, scapula; H, humerus; R, radius; U, ulna. (B) Serial sections of distal humerus were stained with Safranin O and hybridized with indicated 35S labeled riboprobes. The boxed articular and columnar chondrocytes regions are shown at higher magnification in the panels below. Columnar chondrocytes are indicated by yellow brackets and arrows. Both Ptch1c/−;Col2a1-Cre and PTHrP−/−;Ptch1c/−;Col2a1-Cre mutants show strong upregulation of Gli1 and Hip1, downstream target genes of Hh signaling, in the perichondrium and synovial joint. The Col2a1-expressing region (white line) is reduced and the Col10a1-expressing domain (yellow line) is closer to the joint in the PTHrP−/−;Ptch1c/−;Col2a1-Cre mutant.

Article Snippet: Primary antibodies (anti-phospho-Smad1 goat polyclonal IgG (Santa Cruz) at 1:100 and anti-Col2a1 goat polyclonal IgG (Santa Cruz) at 1:200 were used for immunohistochemistry, and signals were detected using the ABC kits (Vector Laboratories) and DAB (Sigma).

Techniques: Staining, Labeling, Expressing, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

doi: 10.1242/dev.018044

Figure Lengend Snippet: (A) Comparison of BrdU-labeled chondrocytes in the cartilage of different mutants and in the wild-type control. Boxed regions are shown at higher magnification in the lower panel. The highly proliferating columnar chondrocytes (bracket) were reduced in the PTHrP−/−;Ptch1c/−;Col2a1-Cre mutant. (B) Percentage of BrdU-labeled chondrocytes in the columnar regions (circled in A), calculated from three independent samples to get the mean±s.d. Student′s t-test, P<0.05.

Article Snippet: Primary antibodies (anti-phospho-Smad1 goat polyclonal IgG (Santa Cruz) at 1:100 and anti-Col2a1 goat polyclonal IgG (Santa Cruz) at 1:200 were used for immunohistochemistry, and signals were detected using the ABC kits (Vector Laboratories) and DAB (Sigma).

Techniques: Labeling, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

doi: 10.1242/dev.018044

Figure Lengend Snippet: (A) Skeletal preparation of E15.5 embryos. Hindlimbs are shown at higher magnifications in the lower panel. (B) Serial sections of tibia were stained with Safranin O and hybridized with 35S labeled Ihh and Col10a1 riboprobes. Smoc/c;Col2a1-Cre mutant tibia showed a slight delay in chondrocyte hypertrophy compared with that of wild-type embryos. PTHrP−/−;Smoc/c;Col2a1-Cre mutant tibia also showed a delay of chondrocyte hypertrophy, as compared with that of the PTHrP−/− mutant. The proliferating chondrocyte region is indicated by the yellow line; the hypertrophic region is indicated by the white line. Fe, femur; T, tibia; Fi, fibula.

Article Snippet: Primary antibodies (anti-phospho-Smad1 goat polyclonal IgG (Santa Cruz) at 1:100 and anti-Col2a1 goat polyclonal IgG (Santa Cruz) at 1:200 were used for immunohistochemistry, and signals were detected using the ABC kits (Vector Laboratories) and DAB (Sigma).

Techniques: Staining, Labeling, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

doi: 10.1242/dev.018044

Figure Lengend Snippet: (A) Serial sections of E14.5 distal humerus were stained by Safranin O and hybridized with a 35S labeled Lef1 probe. Lef1 expression was strongly upregulated in the cartilage of Ptch1c/−;Col2a1-Cre mutants. (B,C) Dual luciferase assay of primary chondrocytes isolated from wild-type newborn pups. Primary chondrocytes were nucleofected with Topflash reporter vectors as a read out for canonical Wnt signaling. Recombinant Shh or Dkk1 protein was added after serum starvation and luciferase activity was measured 24 hours later. Shh treatment or Cre-adenovirus infection of the Ptch1c/c primary chondrocytes activated TOPFLASH activity. Such activation was diminished by Dkk1. (D,E) Quantitative RT-PCR was performed using RNA isolated from primary chondrocytes. Both Axin2 and Lef1 were significantly increased in Shh-treated primary chondrocytes or Cre-adenovirus-infected Ptch1c/c primary chondrocytes compared with untreated samples. Dkk1 treatment blocked the effect of Hh signaling. (F) Immunohistochemistry of E15.5 limb sections (distal ulna) with antibodies against phospho-Smad1, 5 and 8. More phospho-Smad-positive cells were found in the cartilage of the Ptch1c/−;Col2a1-Cre mutant embryos. Boxed region of columnar/prehypertrophic chondrocytes is shown at a higher magnification on the right-hand side. (G) Statistical analysis of phospho-Smad-positive cells in the boxed region of the cartilage. Three samples in the boxed area were counted, and the mean±s.d. are shown. Student′s t-test, P<0.05.

Article Snippet: Primary antibodies (anti-phospho-Smad1 goat polyclonal IgG (Santa Cruz) at 1:100 and anti-Col2a1 goat polyclonal IgG (Santa Cruz) at 1:200 were used for immunohistochemistry, and signals were detected using the ABC kits (Vector Laboratories) and DAB (Sigma).

Techniques: Staining, Labeling, Expressing, Luciferase, Isolation, Recombinant, Activity Assay, Infection, Activation Assay, Quantitative RT-PCR, Immunohistochemistry, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

doi: 10.1242/dev.018044

Figure Lengend Snippet: (A) Skeletal preparations of P15 forelimb and hindlimb of the Ptch1c/c;Col2a1-CreER and wild-type mouse. Unmineralized cartilage in the hand, shoulder and knee joints and the scapula was reduced in the mutant, as shown at high magnification. (Ba-d) Serial sections of distal femur of P12 mice were stained with Safranin O and used for immunohistochemistry to detect Col2a1 expression. There were more hypertrophic chondrocytes with reduced Col2a1 expression in the Ptch1c/c;Col2a1-CreER mouse. (Be-j) Sections of proximal humeri from one-year-old mice were stained with Safranin O. Boxed regions in e and f are shown at higher magnifications in g,h. Cartilage lining was thinner in the Ptch1c/c;Col2a1-CreER mouse (arrow). (i,j) Higher magnification of articular cartilage of the proximal humerus. Joint cartilage in the Ptch1c/c;Col2a1-CreER mouse was significantly reduced (double-headed arrows). (Ca-d) Sections of proximal humeri from 14-month-old mice were stained with Safranin O. Boxed regions in a and b are shown at higher magnifications in c,d. Cartilage lining was thicker in the Smoc/c;Col2a1-CreER mouse (arrow). (Ce,f) Higher magnification of articular cartilage of the proximal humerus. Joint cartilage in the Smoc/c;Col2a1-CreER mouse was significantly thicker (double-headed arrows).

Article Snippet: Primary antibodies (anti-phospho-Smad1 goat polyclonal IgG (Santa Cruz) at 1:100 and anti-Col2a1 goat polyclonal IgG (Santa Cruz) at 1:200 were used for immunohistochemistry, and signals were detected using the ABC kits (Vector Laboratories) and DAB (Sigma).

Techniques: Mutagenesis, Staining, Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Mesenchymal Stem Cell Chondroinduction on Cellulose-Silk Composites is Driven by Substrate Elasticity

doi: 10.1101/383307

Figure Lengend Snippet: Human mesenchymal stem cells were cultured on fibronectin coated composite substrates in stem cell expansion medium supplemented with 10 ng/ml FGF-2. A) Cells were cultured for 14 days after which cells were screened for the expression of the chondrogenic genes - Type II collagen (Col2), Aggrecan (Agg), Sox9 and the marker for dedifferentiation Type I collagen (Col1). Gene expression levels were normalised to the expression of the β -actin housekeeping gene (shown in dotted line). Graph shows mean ± SE, n=4, B) Cells were also cultured for 21 days after which extracellular matrix (ECM) protein deposition was assessed for key cartilage proteins – Type II Collagen (green fluorescence) and Aggrecan (red fluorescence). Cell nuclei were stained using DAPI (blue). Representative images taken at x10 magnification, scale bar inset measures 300 μm.

Article Snippet: Following a further PBS wash step, the samples were incubated with goat anti-human aggrecan antibody (10 μg/ml, R&D Systems), goat anti-human Col2A1 antibody (4 μg/ml, Life Technologies, Paisley, UK) or normal goat IgG (control, 10 μg/ml) overnight at 2-8 °C.

Techniques: Cell Culture, Expressing, Marker, Fluorescence, Staining

Journal: Advanced Science

Article Title: SPP1‐ITGα5/β1 Accelerates Calcification of Nucleus Pulposus Cells by Inhibiting Mitophagy via Ubiquitin‐Dependent PINK1/PARKIN Pathway Blockade

doi: 10.1002/advs.202411162

Figure Lengend Snippet: Increased SPP1 expression in degenerative intervertebral discs synchronized with calcification and disturbance of extracellular mechanism. A) ARS staining, 500 µm, n = 5; B) Safranin O staining, 500 µm, n = 5; C) Representative immunofluorescence images of COL1A1 and SPP1, 500 µm, 20 µm, n = 5; D) Representative immunofluorescence images of COL2A1 and MMP13, 500 µm, 20 µm, n = 5; E) Relative mRNA level of SPP1, COL1A1, BGLAP, COL2A1, ACAN, MMP13, ADAMTS5, n = 5. * p < 0.05, ** p < 0.01 versus Pfirrmann II.

Article Snippet: The antibodies of COL1A1 (A16891), COL2A1 (A19308), MMP13 (A11755), SPP1 (A1361), goat anti‐rabbit IgG (AC005), FITC goat anti‐rabbit IgG (H+L) (AS011) and cy3 goat anti‐rabbit IgG (H+L) (AS007) were purchased from ABclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing, Staining, Immunofluorescence

Journal: Advanced Science

Article Title: SPP1‐ITGα5/β1 Accelerates Calcification of Nucleus Pulposus Cells by Inhibiting Mitophagy via Ubiquitin‐Dependent PINK1/PARKIN Pathway Blockade

doi: 10.1002/advs.202411162

Figure Lengend Snippet: Increased SPP1 was involved in degenerative intervertebral discs of rats. A) Safranin O staining, 500 µm, n = 5; B) Representative immunofluorescence images of COL1A1 and SPP1, 250 µm, n = 5; C) Representative immunofluorescence images of COL2A1 and MMP13, 250 µm, n = 5; D) Relative mRNA level of SPP1, COL1A1, BGLAP, COL2A1, ACAN, MMP13, ADAMTS5, n = 5. * p < 0.05, ** p < 0.01 versus Sham.

Article Snippet: The antibodies of COL1A1 (A16891), COL2A1 (A19308), MMP13 (A11755), SPP1 (A1361), goat anti‐rabbit IgG (AC005), FITC goat anti‐rabbit IgG (H+L) (AS011) and cy3 goat anti‐rabbit IgG (H+L) (AS007) were purchased from ABclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Staining, Immunofluorescence

Journal: Advanced Science

Article Title: SPP1‐ITGα5/β1 Accelerates Calcification of Nucleus Pulposus Cells by Inhibiting Mitophagy via Ubiquitin‐Dependent PINK1/PARKIN Pathway Blockade

doi: 10.1002/advs.202411162

Figure Lengend Snippet: SPP1 promoted osteogenic differentiation of nucleus pulposus cells and disrupted extracellular matrix homeostasis under inflammatory conditions. A,B) Apoptosis flow cytometry, n = 3; C) Relative mRNA level of COL1A1 and BGLAP, n = 6; D,E) Representative immunofluorescence images of COL1A1, 250 µm, n = 3; F,G) ALP staining, ARS staining, Safranin O staining and alcian staining, 200×, n = 3; H,I) Representative immunofluorescence images of COL2A1 and MMP13, 200 µm, n = 3; J) Relative mRNA level of ACAN, COL2A1, MMP13 and ADAMTS5, n = 6. * p < 0.05, ** p < 0.01 versus IL‐1β+NC‐siRNA.

Article Snippet: The antibodies of COL1A1 (A16891), COL2A1 (A19308), MMP13 (A11755), SPP1 (A1361), goat anti‐rabbit IgG (AC005), FITC goat anti‐rabbit IgG (H+L) (AS011) and cy3 goat anti‐rabbit IgG (H+L) (AS007) were purchased from ABclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Flow Cytometry, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: SPP1‐ITGα5/β1 Accelerates Calcification of Nucleus Pulposus Cells by Inhibiting Mitophagy via Ubiquitin‐Dependent PINK1/PARKIN Pathway Blockade

doi: 10.1002/advs.202411162

Figure Lengend Snippet: SPP1 knockdown attenuated matrix disturbance and calcification tendency by mitophagy activation in intervertebral discs. A) Safranin O staining, 500 µm, n = 5; B) Representative immunofluorescence images of COL1A1, 250 µm, n = 5; C) Representative immunofluorescence images of COL2A1 and MMP13, 250 µm, n = 5; D) Representative immunofluorescence images of PINK1 and PARKIN, 250 µm, n = 5; E) Representative TEM images of the rat nucleus pulposus tissue, 1 µm, n = 3; F) Relative mRNA level of SPP1, BGLAP, CLO1A1, ACAN, COL2A1, MMP13, ADAMTS5, SQSTM1, LC3B, ATG5, LAMP1, BECN1, PINK1, and PARKIN, n = 6. * p < 0.05, ** p < 0.01 versus IVDD+NC‐siRNA.

Article Snippet: The antibodies of COL1A1 (A16891), COL2A1 (A19308), MMP13 (A11755), SPP1 (A1361), goat anti‐rabbit IgG (AC005), FITC goat anti‐rabbit IgG (H+L) (AS011) and cy3 goat anti‐rabbit IgG (H+L) (AS007) were purchased from ABclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Knockdown, Activation Assay, Staining, Immunofluorescence

Journal: Advanced Science

Article Title: SPP1‐ITGα5/β1 Accelerates Calcification of Nucleus Pulposus Cells by Inhibiting Mitophagy via Ubiquitin‐Dependent PINK1/PARKIN Pathway Blockade

doi: 10.1002/advs.202411162

Figure Lengend Snippet: SPP1‐ITGα5/β1 signaling promoted osteogenesis and extracellular matrix homeostasis disruption of NPs. A) Protein interaction network analysis; B) Relative mRNA level of ITGαV, ITGα5, ITGβ1, ITGβ3 and ITGβ5 of NPs, n = 6; C,D) Representative immunofluorescence images of SPP1 and ITGα5 of NPs, 250 µm, n = 3; E,F) Representative immunofluorescence images of SPP1 and ITGα5 of nucleus pulposus tissue (Pfirrmann II and Pfirrmann IV), 500 µm, 200 µm, n = 3; G) Co‐immunoprecipitation assays, n = 3; H) Relative mRNA level of ITGαV, ITGα5, ITGβ1, ITGβ3, and ITGβ5 of nucleus pulposus tissue (Pfirrmann II and Pfirrmann IV), n = 5; I,J) Flow cytometry of apoptosis, n = 3; K) Relative mRNA level of COL1A1, BGLAP, ACAN, COL2A1, MMP13, and ADAMTS5, n = 6; L,M) Representative immunofluorescence images of COL1A1, 400×, n = 3; N,O) Representative immunofluorescence images of COL2A1 and MMP13, 630×, n = 3; P,Q) ALP staining, ARS staining, Safranin O staining and alcian staining, 200×, n = 3; R) ITGα5 induced senescence through ROS accumulation mediated by mitophagy reduction in NPs, the picture was drawn by Biorender ( https://app.biorender.com/ ). Means ± S.E.M. * p < 0.05, ** p < 0.01 versus IL‐1β+NC‐siRNA & versus Pfirrmann II & AAV‐shNC, # p < 0.05, ## p < 0.01 versus IL‐1β+si‐SPP1 & versus IL‐1β+AAV‐shITGα5.

Article Snippet: The antibodies of COL1A1 (A16891), COL2A1 (A19308), MMP13 (A11755), SPP1 (A1361), goat anti‐rabbit IgG (AC005), FITC goat anti‐rabbit IgG (H+L) (AS011) and cy3 goat anti‐rabbit IgG (H+L) (AS007) were purchased from ABclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Disruption, Immunofluorescence, Immunoprecipitation, Flow Cytometry, Staining

Journal: Advanced Science

Article Title: SPP1‐ITGα5/β1 Accelerates Calcification of Nucleus Pulposus Cells by Inhibiting Mitophagy via Ubiquitin‐Dependent PINK1/PARKIN Pathway Blockade

doi: 10.1002/advs.202411162

Figure Lengend Snippet: ITGα5 knockdown attenuated matrix disturbance and calcification tendency by mitophagy activation in intervertebral discs. A) Safranin O staining, 100×, n = 6; C) Relative mRNA level of BGLAP, CLO1A1, ACAN, COL2A1, MMP13, and ADAMTS5, n = 6; B) Representative immunofluorescence images of COL1A1, COL2A1 and MMP13, PINK1, and PARKIN, 400×, n = 3; (D) Representative TEM images of the rat nucleus pulposus tissue, 14 000×, n = 3; E) Relative mRNA level of SQSTM1, LC3B, ATG5, LAMP1, BECN1, PINK1 and PARKIN, n = 6. * p < 0.05, ** p < 0.01 versus IVDD+AAV‐shNC.

Article Snippet: The antibodies of COL1A1 (A16891), COL2A1 (A19308), MMP13 (A11755), SPP1 (A1361), goat anti‐rabbit IgG (AC005), FITC goat anti‐rabbit IgG (H+L) (AS011) and cy3 goat anti‐rabbit IgG (H+L) (AS007) were purchased from ABclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Knockdown, Activation Assay, Staining, Immunofluorescence