Journal: International Journal of Molecular Sciences

Article Title: ER Stress in ERp57 Knockout Knee Joint Chondrocytes Induces Osteoarthritic Cartilage Degradation and Osteophyte Formation

doi: 10.3390/ijms23010182

Figure Lengend Snippet: ERp57 cKO mice show an accelerated development of osteoarthritis during aging. ( A ) Histological analysis and OARSI scoring of toluidine blue-stained knee sections of 18-month-old mice reveals an increase in cartilage degradation, the occurrence of hypertrophic chondrocytes (*), and osteophyte formation (scattered box) in ERp57 cKO mice, but not in WT animals ( p = 0.0039). No significant difference was detected between younger WT and ERp57 cKO mice at the age of 9 months ( p = 0.7924) and 12 months ( p = 0.2503). Scale Bars = 100 µm. ( B ) TUNEL staining indicates a higher percentage of apoptotic cells in the articular cartilage of ERp57 cKO mice at 9 ( p = 0.0271), 12 ( p = 0.0490) and 18 months ( p = 0,000000028) compared to WT animals. Scale bars = 50 µm ( C ) Immunofluorescence of 18-month-old ERp57 cKO cartilage reveals increased staining intensities of matrix degrading proteases MMP3 ( p = 0.0004), MMP9 ( p = 0.0097), ADAMTS4 ( p = 0.0428), and ADAMTS5 ( p = 0.0288) compared to WT controls. M = months. n ≥ 4. *** p < 0.005, ** p < 0.01, * p < 0.05.

Article Snippet: Then the Vectastain ® Elite ABC Universal Kit (VEC-PK-7200, BIOZOL, Eching, Germany) and the Vector ® DAB Peroxidase Substrate (VEC-SK-4100, BIOZOL, Eching, Germany) were used according to the manufacturer’s instructions with polyclonal antibodies (anti-ADAMTS4: bs-4191R, Bioss Antibodies Inc., Woburn, MA, USA, 1:200 and ADAMTS5: bs-3573R, Bioss Antibodies Inc, Woburn, MA, USA, 1:50), which were incubated on the slides overnight at 4 °C.

Techniques: Staining, TUNEL Assay, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: ER Stress in ERp57 Knockout Knee Joint Chondrocytes Induces Osteoarthritic Cartilage Degradation and Osteophyte Formation

doi: 10.3390/ijms23010182

Figure Lengend Snippet: ER stress-induced osteoarthritic changes in the knee joint. Knee joints of aged ERp57 cKO animals display severe OA cartilage degeneration with enhanced MMP3, MMP9, ADAMTS4, and ADAMTS5 expression and osteophyte formation. These changes are preceded by reduced compressive stiffness of the cartilage ECM and increased apoptotic cell death. Both effects are likely induced by ER stress that cannot be adequately compensated by the UPR.

Article Snippet: Then the Vectastain ® Elite ABC Universal Kit (VEC-PK-7200, BIOZOL, Eching, Germany) and the Vector ® DAB Peroxidase Substrate (VEC-SK-4100, BIOZOL, Eching, Germany) were used according to the manufacturer’s instructions with polyclonal antibodies (anti-ADAMTS4: bs-4191R, Bioss Antibodies Inc., Woburn, MA, USA, 1:200 and ADAMTS5: bs-3573R, Bioss Antibodies Inc, Woburn, MA, USA, 1:50), which were incubated on the slides overnight at 4 °C.

Techniques: Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Flavokawain A alleviates the progression of mouse osteoarthritis: An in vitro and in vivo study

doi: 10.3389/fbioe.2022.1071776

Figure Lengend Snippet: Protective effects of FKA against IL-1β-induced inflammation and ECM destruction in mouse chondrocytes. Chondrocyte cell treatment with 5 ng/ml of IL-1β only or with FKA (20 and 40 μM) for a period of 24 h. Western blot analysis of inflammatory cytokines (COX2 and iNOS), catabolic (ADAMTS5, MMP3, and MMP13), and anabolic markers (Col2, Aggrecan, and Sox9) (A,C and E) . Quantitative analysis of protein expression (B,D and F) . (G and H) 5 ng/ml of IL-1β was added for treating the cells for 24 h in the absence or presence of 40 µM FKA. Confocal microscopy showing the expression of MMP13 and Aggrecan in immunofluorescence experiments (scale bar 10 μm). (I) Relative quantification of fluorescence intensity. The values are presented as means ± SD ( n = 3). # p < 0.05 vs the control group; * p < 0.05 and ** p < 0.01 vs the IL-1β group.

Article Snippet: ADAMTS5 primary antibody, secondary antibody, phosphate-buffered saline (PBS), trypsin, collagenase type II, the CCK8 assay kit, bovine serum albumin (BSA), and protein extraction kit were ordered and acquired from Boster Biological Technology (Wuhan, Hubei, China).

Techniques: Western Blot, Expressing, Confocal Microscopy, Immunofluorescence, Quantitative Proteomics, Fluorescence, Control

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