Journal: Cartilage

Article Title: HSF1 Increases EOGT-Mediated Glycosylation of Notch1 to Promote IL-1β-Induced Inflammatory Injury of Chondrocytes.

doi: 10.1177/19476035241229211

Figure Lengend Snippet: Figure 2. EOGT knockdown alleviated IL-1β-induced chondrocyte inflammatory injury. EOGT knockdown was induced in IL-1β- treated chondrocytes. (A) IL-6 and IL-8 secretion levels in chondrocytes were assessed by ELISA. (B-C) IF was adopted to detect MMP13 and ADAMTS5 fluorescence intensity in chondrocytes. Data were expressed as mean ± SD. All our data were obtained from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. EOGT = epidermal growth factor–like domain–specific O-GlcNAc transferase; IL-1β = interleukin-1 beta; ELISA = enzyme-linked immunosorbent assay.

Article Snippet: Cells were subsequently incubated overnight with antibodies against MMP13 (Proteintech, Wuhan, China; 1:500, 18165-1-AP) and AdAMTS5 (ABclonal, Wuhan, China; 1:200, A2836) for 2 h. Then, cells were incubated with the secondary antibody (Abcam; 1:3000, ab7090) for 1 h, and the nucleus was stained with DAPI (Sangon).

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: Cartilage

Article Title: HSF1 Increases EOGT-Mediated Glycosylation of Notch1 to Promote IL-1β-Induced Inflammatory Injury of Chondrocytes.

doi: 10.1177/19476035241229211

Figure Lengend Snippet: Figure 3. HSF1 was highly expressed in IL-1β-treated chondrocytes and was involved in Notch1-mediated inflammatory injury. (A-B) HSF1 expression level in human chondrocytes after IL-1β stimulation was measured by RT-qPCR and western blot. (C-D) RT-qPCR and western blot were adopted to examine HSF1 expression level in human chondrocytes following si-NC or si-HSF1 transfection. HSF1 knockdown was induced in IL-1β-treated chondrocytes. (E-F) HSF1 expression in chondrocytes was measured by RT-qPCR and western blot. (G) Notch1 and NICD1 protein levels in cells were examined using western blot. (H) ELISA was adopted to detect IL-6 and IL-8 secretion levels in chondrocytes. (I-J) IF was adopted to detect MMP13 and ADAMTS5 fluorescence intensity in cells. Data were expressed as mean ± SD. All our data were obtained from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. HSF1 = heat-shock transcription factor 1; EOGT = epidermal growth factor–like domain–specific O-GlcNAc transferase; IL-1β = interleukin-1 beta; RT-qPCR = reverse transcription-quantitative polymerase chain reaction; ELISA = enzyme-linked immunosorbent assay; IF = immunofluorescence.

Article Snippet: Cells were subsequently incubated overnight with antibodies against MMP13 (Proteintech, Wuhan, China; 1:500, 18165-1-AP) and AdAMTS5 (ABclonal, Wuhan, China; 1:200, A2836) for 2 h. Then, cells were incubated with the secondary antibody (Abcam; 1:3000, ab7090) for 1 h, and the nucleus was stained with DAPI (Sangon).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Knockdown, Enzyme-linked Immunosorbent Assay, Fluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Enhancing autophagy and energy metabolism in the meniscus can delay the occurrence of PTOA in ACLT rat

doi: 10.3389/fcell.2022.971736

Figure Lengend Snippet: Influence of the meniscus on articular cartilage during the development of PTOA (A) . The immunofluorescence staining results of MMP13 in 1 week, 2 and 3 weeks after ACLT surgery (Scale bar: the left panel was 100 um, the middle and the right panel was 25 um). (B) The statistics of MMP13-positive cells in tibia (C) . The statistics of MMP13-positive cells in meniscus (D) . The expression of Adamts5 and MMP13 in Western Blot (E) . The gray value statistics of Adamts5 (F) The gray value statistics of MMP13.

Article Snippet: HE staining solution, Toluidine blue staining powder and safranin O/fast green staining powder was buy from Sigma (United States), SDS-PAGE gel rapid preparation kit was attained from Beyotime Biotechnology (Shanghai)Co., Ltd. rapamycin was acquired from Shanghai yuanye Bio-Technology Co., Ltd., IL-1β was purchased from R&D Systems (Bio-Techne), 3-methyladenine (3-MA, autophagy inhibitor) was produced from TargetMol (United States), primary antibodies MMP13 (18165-1-AP), Adamts5 (Ag26730), P-AKT (Phospho-AKT1 (Ser473)), Beclin 1 (11306-1-AP), LC-3B (18725-1-AP), ULK1 (20986-1-AP), ATG12 (11264-1-AP), COL1 (66761-1-lg) achieved from Proteintech Group, COL2 (GB11021) were procured from Servicebio (China), P-S6K (9208S)/S6K (9202S) were come from Cell Signaling Technology (CST), P62 (ab109012) was purchased from Abcam (Shanghai).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ADAMTS5 Deficiency in Calcified Aortic Valves Is Associated With Elevated Pro-Osteogenic Activity in Valvular Interstitial Cells

doi: 10.1161/atvbaha.117.309021

Figure Lengend Snippet: Figure 7. ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) knockdown enhances the effect of matrilin 2 on valvular interstitial cell (VIC) phenotypic change. A, VICs from calcified aortic valves were treated with recombinant human ADAMTS5 (rhADAMTS5; 5.0 µg/mL) for 48 h. Immunoblots and densitometric data show that ADAMTS5 decreased the baseline levels of α-SMA (α-smooth muscle actin), Runx2 (runt-related transcription factor 2), and ALP (alkaline phosphatase). B, VICs from normal aortic valves were treated with rhADAMTS5 (5.0 µg/mL) 1 h before matrilin 2 (2.0 μg/mL) stimulation for 48 h. ADAMTS5 attenuated the upregulation of α-SMA, Runx2, and ALP by matrilin 2. C, ADAMTS5 small interfering RNA (siRNA) reduced ADAMTS5 levels in VICs from normal aortic valves. D and E, VICs from normal aortic valves were transfected with ADAMTS5 siRNA 72 h before matrilin 2 stimulation for 48 h. Immu- noblots and densitometric data show that ADAMTS5 knockdown augmented the upregulation of α-SMA, Runx2, and ALP by matrilin 2. Mean±SE; n=4 separate experiments using distinct cell isolates; *P<0.05 vs untreated control and control siRNA-treated; #P<0.05 vs matrilin 2 alone or matrilin 2+control siRNA.

Article Snippet: Recombinant human ADAMTS5 and matrilin2 proteins (expressed by mouse myeloma cell line; endotoxin free) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Knockdown, Recombinant, Western Blot, Small Interfering RNA, Transfection, Control

Journal: Cartilage

Article Title: Ferroptosis Plays a Role in Human Chondrocyte of Osteoarthritis Induced by IL-1β In Vitro .

doi: 10.1177/19476035221142011

Figure Lengend Snippet: Figure 6. Fer-1 reduced the secretion of OA markers of chondrocyte. (A) The protein expression level of ADAMTS5 and MMP- 13 of each group detected through Western blot analysis. (B) The band density ratio of ADAMTS5 and MMP-13 to β-actin in the Western blot quantified by densitometry. (C) Viability of chondrocytes treated with different concentrations of Lip-1. (D-E) relative expression of mRNAs related to ferroptosis and OA inflammation in different groups. (F) The level of ADAMTS5 concentration of different groups in the cell supernatant. (G) The level of MMP-13 concentration of different groups in the cell supernatant. OA = osteoarthritis; ADAMTS5 = a disintegrin and metalloproteinase with thrombospondinmotif-5; MMP-13 = matrix metallopeptidase 13; IL-1β = interleukin-1β; GPX4 = glutathione peroxidase; ACSL4 = acyl-CoA synthetase long-chain family member 4. *P < 0.05, ***P < 0.001, ****P < 0.0001. ns = no significance.

Article Snippet: Images were obtained under a fluorescence microscope. eliSA Analysis Chondrocyte supernatant was collected and centrifuged for 20 minutes at 1,000 × g at 2°C to 8°C, then was added to a 96-well plate covered by antibody of ADAMTS5 (Elabscience, USA, E-EL-H5590c) and MMP-13 (Elabscience, USA, E-EL-H6023), 100 μl for each well.

Techniques: Expressing, Western Blot, Concentration Assay