Journal: Cartilage

Article Title: Cellular Senescence in Human Chondrocytes in Relation to Osteoarthritis

doi: 10.1177/19476035251344875

Figure Lengend Snippet: Study design for analyzing chondrocytes for cellular senescence.

Article Snippet: The human primary chondrocyte cell line was purchased from iCell Biotech Co.

Techniques:

Journal: Cartilage

Article Title: Cellular Senescence in Human Chondrocytes in Relation to Osteoarthritis

doi: 10.1177/19476035251344875

Figure Lengend Snippet: Bleomycin-induced cellular senescence and apoptosis. ( A ) Representative images of SA-β-gal in chondrocytes treated with bleomycin for 2 hours. Scale bar = 100 μm. ( B ) Representative images of SA-β-gal in chondrocytes treated with bleomycin for 2 hours and incubated for 7 days. Scale bar = 100 μm. ( C ) SA-β-gal positive rate for each group. Images from 10 nonoverlapping fields were collected per group for quantification. ( D, E ) p16 and p21 expression level for each group. Each group included six replicates. ( F ) The ratio of Bcl-2 expression level to Bax expression level for each group. Each group included six replicates. Data are shown as average ± SD.

Article Snippet: The human primary chondrocyte cell line was purchased from iCell Biotech Co.

Techniques: Incubation, Expressing

Journal: Cartilage

Article Title: Cellular Senescence in Human Chondrocytes in Relation to Osteoarthritis

doi: 10.1177/19476035251344875

Figure Lengend Snippet: ENU does not induce cellular senescence or apoptosis. ( A ) Representative images of SA-β-gal in chondrocytes treated with ENU. Scale bar = 100 μm. ( B ) SA-β-gal positive rate for three groups. Images from 10 nonoverlapping fields were collected per group for quantification. ( C, D ) p16 and p21 expression level for three groups. Each group included nine replicates. ( E ) The ratio of Bcl-2 expression level to Bax expression level for three groups. Each group included nine replicates. Data are shown as average ± SD.

Article Snippet: The human primary chondrocyte cell line was purchased from iCell Biotech Co.

Techniques: Expressing

Journal: Cartilage

Article Title: Cellular Senescence in Human Chondrocytes in Relation to Osteoarthritis

doi: 10.1177/19476035251344875

Figure Lengend Snippet: Cellular senescence and apoptosis biomarkers in chondrocytes from articular cartilage. ( A-I ) Age-associated trends and intergroup comparison in p16 and p21 expression levels and the ratio of Bcl-2 expression level to Bax expression level. Each sample included three replicates. Data are shown as boxplots indicating the interquartile range (IQR), with the center line representing the median and whiskers extending to 1.5 × IQR. ( J , K ). SA-β-gal and TUNEL positive rates for each group. The rates were calculated by dividing the number of positive cells by the total number of cells. Each group included three replicates. Data are shown as average ± SD.

Article Snippet: The human primary chondrocyte cell line was purchased from iCell Biotech Co.

Techniques: Comparison, Expressing, TUNEL Assay

Journal: Aging (Albany NY)

Article Title: Overexpression of miR-10a-5p facilitates the progression of osteoarthritis

doi: 10.18632/aging.102989

Figure Lengend Snippet: HOXA3 acts as a targeted gene of miR-10a-5p. ( A ) Volcano plot of differentially expressed mRNAs between control group and IL-1β induced primary chondrocyte group from GSE74220. ( B ) Venn plot of the overlapping mRNA across the downregulated mRNAs in GSE74220, the predicted targets of miR-10a-5p in Targetscan, and the downregulated mRNAs in our RNA-seq. ( C ) The expression level of HOXA3 in OA cartilage from GSE114007, **P < 0.01. ( D ) Targetscan shows that the seed region of miR-10a-5p is well-paired with 3’UTR of HOXA3, which was conservative among vertebrates. ( E ) High-throughput experiments verified that HOXA3 was the direct target of miR-10a-5p in HEK293, TZMBL, and 293S from TarBase v.8. ( F ) The sequencing results of cloned fragments in luciferase reporter vectors and the yellow highlighted sequence is the target test site. ( G ) Interaction between miR-10a-5p and HOXA3 was verified by luciferase report assay in 293T cells. ( H , I ) The relative expression of HOXA3 in control and IL-1β induced Hc-a analyzed by RT-qPCR and western blot (n=3, *P < 0.05). ( J , K ) The relative expression of HOXA3 after transfecting miR-10a-5p mimics analyzed by RT-qPCR and western blot (n=3, **P < 0.01).

Article Snippet: Human primary chondrocytes (HC-a; Catalog #4650) was purchased from ScienCell Research Laboratories.

Techniques: Control, RNA Sequencing, Expressing, High Throughput Screening Assay, Sequencing, Clone Assay, Luciferase, Quantitative RT-PCR, Western Blot

Journal: Aging (Albany NY)

Article Title: Overexpression of miR-10a-5p facilitates the progression of osteoarthritis

doi: 10.18632/aging.102989

Figure Lengend Snippet: Silence of HOXA3 inhibited chondrocyte proliferation and promoted chondrocyte apoptosis. ( A ) The relative expression of HOXA3 after transfecting si-HOXA3 analyzed by RT-qPCR (n=3; ***P < 0.001). ( B ) The relative expression of HOXA3 after transfecting si-HOXA3 analyzed by western blot. ( C ) The effect of HOXA3 knockdown on cell proliferation detected by CCK8 assay (n=3; *P < 0.05, **P < 0.01). ( D , F ) The effect of HOXA3 knockdown on cell proliferation detected by EDU assay (n=3; *P < 0.05). ( E , G ) The effect of HOXA3 knockdown on cell apoptosis detected by flow cytometry assay (n=3; *P < 0.05). ( H ) Effects of HOXA3 knockdown on Col2a1, MMP13, BAX, and GAPDH protein levels detected by western blot.

Article Snippet: Human primary chondrocytes (HC-a; Catalog #4650) was purchased from ScienCell Research Laboratories.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, EdU Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Single-cell RNA sequencing reveals different chondrocyte states in femoral cartilage between osteoarthritis and healthy individuals

doi: 10.3389/fimmu.2024.1407679

Figure Lengend Snippet: Differentiation sequence of chondrocyte subtypes (A, D, G) pseudo-time analysis of all cells, NC group and OA group. The dots in each color represents each state, and each state is named numerically. The direction of the arrow represents the direction of the pseudo-time. (B, E, H) umap visualization of state in (A, D, G) . The meanings of the colors, numbers, and arrows correspond to the (A, D, G) , respectively. (C, F, I, J) RNA velocity analysis of EC-A, EC-B, and RegC-B in all cells, NC group, and OA group cells. (K) Relative expression levels of COL1/2 subtypes (L) Summary of the differentiation sequence of each subtype. The arrows represent the differentiation direction.

Article Snippet: The immortalized human chondrocytes (iCell Bioscience Inc, Shanghai) were passaged onto glass slides in a 6-well plate and grown to an experimental usable state.

Techniques: Sequencing, Expressing

Journal: Frontiers in Immunology

Article Title: Single-cell RNA sequencing reveals different chondrocyte states in femoral cartilage between osteoarthritis and healthy individuals

doi: 10.3389/fimmu.2024.1407679

Figure Lengend Snippet: Transcriptional regulation of chondrocyte subtypes (A) Heatmap of specific transcriptional factors of EC-A, EC-B, and RegC-B (B) RSS scores for specific transcription factors of EC-A, EC-B and RegC-B. (C–E) umap plot for key transcription factors of EC-A, EC-B, and RegC-B. (F) Gene regulatory network of specific transcription factors. The purple genes are the screened key transcription factor. The green and yellow genes are the highly expressed DEGs in the chondrocyte subtypes, and the yellow genes means that the gene itself is a transcription factor.

Article Snippet: The immortalized human chondrocytes (iCell Bioscience Inc, Shanghai) were passaged onto glass slides in a 6-well plate and grown to an experimental usable state.

Techniques:

Journal: Polymers

Article Title: Bioactive Hydrogel Scaffolds Integrating Chitosan, Silk Fibroin, and Aloe vera Extract for Enhanced Cartilage Tissue Regeneration

doi: 10.3390/polym17101409

Figure Lengend Snippet: Chondrocyte viability assessment via MTT assay comparing conventional monolayer culture (2D) and CS40/SF/AV scaffold encapsulation (3D) at days 1 and 7. Measurements show formazan absorbance at 540 nm, with bars representing mean ± S.D. from triplicate experiments ( n = 3). Statistical analysis revealed significant differences between day 1 and day 7 within each culture system (** p < 0.01, independent t -test). Additionally, two-way ANOVA indicated significant differences between the 2D and 3D culture environments (** p < 0.01).

Article Snippet: Cells were maintained in the Primary Chondrocyte Culture System (PriMed-iCell-020, iCell) at 37 °C under 5% CO 2 atmospheric conditions.

Techniques: MTT Assay, Encapsulation

Journal: Polymers

Article Title: Bioactive Hydrogel Scaffolds Integrating Chitosan, Silk Fibroin, and Aloe vera Extract for Enhanced Cartilage Tissue Regeneration

doi: 10.3390/polym17101409

Figure Lengend Snippet: Cell morphology imaged by inverted microscopy: cell suspension culture ( a ), and chondrocytes cultured on the tissue culture plate (2D culture) for 7 days ( b ); SEM micrograph of CS40/SF/AV scaffold without seeding cell culture ( c ); SEM micrograph of chondrocyte cultured on the CS40/SF/AV scaffold for 7 days (3D culture, ( d – f )), where white arrows indicate individual chondrocyte cells attached to and integrated with scaffold structure; and fluorescence microscopy image of chondrocyte nuclei stained with DAPI after 28 days of culture on CS40/SF/AV scaffold ( g ).

Article Snippet: Cells were maintained in the Primary Chondrocyte Culture System (PriMed-iCell-020, iCell) at 37 °C under 5% CO 2 atmospheric conditions.

Techniques: Inverted Microscopy, Suspension, Cell Culture, Fluorescence, Microscopy, Staining

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: Atf3 expression is upregulated during chondrocyte differentiation in vitro . Atf3 mRNA expression during chondrogenic differentiation of mouse embryonic limb bud cells in micromass culture was examined by microarray (A) and real-time PCR (B) analyses (different cell isolations were used for the experiments in A and B). Microarray data sets represent averages from three independent experiments. Real-time data were normalized to Gapdh levels and present average and SEM from four independent experiments. Both approaches show similar expression patterns with a strong increase in Atf3 expression during micromass differentiation.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Expressing, In Vitro, Microarray, Real-time Polymerase Chain Reaction

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: Atf3 expression is upregulated during chondrocyte differentiation in vivo . Tibia isolated from embryonic day 15.5. mice were microdissected into the resting/proliferating, prehypertrophic and hypertrophic areas, and RNA was isolated directly out of cartilage. Expression of type II collagen II ( Col2a1 ), type X collagen ( Col10a1 ) and Atf3 was examined by real-time PCR analyses and normalized to Gapdh expression. All data represent averages and SEM from four independent experiments (*: p < 0.05). Col2a1 was strongly expressed in the resting/proliferating area and declined subsequently (A). Col10a1 was virtually undetectable in the resting/proliferating area and strongly induced in the prehypertrophic and hypertrophic areas (B). Atf3 was already expressed in the resting/proliferating area, but expression was significantly increased in the more mature zones of the growth plate.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Expressing, In Vivo, Isolation, Real-time Polymerase Chain Reaction

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: Inhibition of actin polymerization induces Atf3 expression through transcriptional mechanisms . A) Primary chondrocytes were cultured for 24 hours with DMSO (control) or 1 μM cytochalasin D before harvest and RNA isolation. Real-time PCR demonstrated marked induction of Atf3 mRNA levels by cytochalasin D. Data represent averages and SEM from three independent experiments, normalized to Gapdh (*: p < 0.05). B) Primary chondrocytes were transfected with an Atf3 promoter plasmid and pRLCMV, followed by incubation for 24 hours with DMSO (control) or 1 μM cytochalasin D. Cells were then harvested, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data represent averages and SEM from three independent experiments, performed in quadruplicate each (*: p < 0.05). Cytochalasin D induced Atf3 promoter activity.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Inhibition, Expressing, Cell Culture, Control, Isolation, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: ATF3 suppresses Sox9-dependent transcription and stimulates Runx2-dependent transcription . Primary chondrocytes were cotransfected with SOX9 (A) or RUNX2 (B) reporter plasmids, pcDNA3 (control) or an ATF3 expression vector, and pRLCMV. 24 hours after transfection, cells were harvested, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data represent averages and SEM from three independent experiments, performed in quadruplicate each (*: p < 0.05). ATF3 overexpression suppressed SOX9-dependent transcription and stimulated RUNX2-dependent transcription.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Control, Expressing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Over Expression

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: ATF3 suppresses CRE-dependent transcription . Primary chondrocytes were cotransfected with a CRE reporter (A), a cyclin D1 promoter (-1745CD1LUC, B) or cyclin A promoter plasmids (p707cycAluc and p707cycAlucMut; C), pcDNA3 (control) or an ATF3 expression vector, and pRLCMV. 24 hours after transfection, cells were harvested, firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data represent averages and SEM from three independent experiments, performed in quadruplicate each (*: p < 0.05). ATF3 overexpression suppressed the CRE reporter, cyclin D1 and cyclin A wild type promoters significantly, whereas the mutant cyclin A construct showed no response to ATF3 overexpression.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Control, Expressing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Over Expression, Mutagenesis, Construct

Journal: BMC Molecular Biology

Article Title: The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

doi: 10.1186/1471-2199-7-30

Figure Lengend Snippet: Model for ATF3 action in chondrocyte differentiation . We suggest that induction of ATF3 by differentiation stimuli antagonizes the CRE-dependent expression of cyclin D1 and cyclin A, which are induced by mitogenic stimuli through ATF2 and CREB. Reduced cyclin-dependent kinase activity and hypophosphorylation of pocket proteins in response to ATF3 upregulation then results both in cell cycle exit and in increased activity of Runx2, promoting chondrocyte differentiation.

Article Snippet: Micromass cultures from E11.5 mouse limb buds and isolation of primary murine chondrocytes from E15.5 long bones were performed as recently described [ , ].

Techniques: Expressing, Activity Assay