Journal: Arthritis Research & Therapy

Article Title: Excessive mechanical stress-induced intervertebral disc degeneration is related to Piezo1 overexpression triggering the imbalance of autophagy/apoptosis in human nucleus pulpous

doi: 10.1186/s13075-022-02804-y

Figure Lengend Snippet: Primers for real-time PCR

Article Snippet: After deparaffinization, rehydration, antigen retrieval, and blocking, the slides were incubated with primary antibodies against Piezo1 (No. NBP1-78537, Novus Biologicals, USA, 1:100 dilution) and type II Collagen (No. NBP1-77795, Novus Biologicals, USA, 1:200 dilution) on a humidified box at 4 °C overnight.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Excessive mechanical stress-induced intervertebral disc degeneration is related to Piezo1 overexpression triggering the imbalance of autophagy/apoptosis in human nucleus pulpous

doi: 10.1186/s13075-022-02804-y

Figure Lengend Snippet: Histological analysis and the expression of Piezo1 and type II collage in human nucleus pulposus tissues. A , B Hematoxylin and eosin (H&E) staining in human NP tissues of the control or the IVDD group (200×). C , D Piezo1 immunostaining in human NP tissues of the control or the IVDD group (100×). E The percentage of Piezo1 positive cells to all cells in the field (%). F , G Type II collage immunostaining in human NP tissues of the control or the IVDD group (100×). H The percentage of Col II positive cells to all cells in the field (%). * p < 0.05

Article Snippet: After deparaffinization, rehydration, antigen retrieval, and blocking, the slides were incubated with primary antibodies against Piezo1 (No. NBP1-78537, Novus Biologicals, USA, 1:100 dilution) and type II Collagen (No. NBP1-77795, Novus Biologicals, USA, 1:200 dilution) on a humidified box at 4 °C overnight.

Techniques: Expressing, Staining, Control, Immunostaining

Journal: Arthritis Research & Therapy

Article Title: Excessive mechanical stress-induced intervertebral disc degeneration is related to Piezo1 overexpression triggering the imbalance of autophagy/apoptosis in human nucleus pulpous

doi: 10.1186/s13075-022-02804-y

Figure Lengend Snippet: Piezo1 expression and cell function of human nucleus pulposus cells under mechanical stress. A NP cells received various mechanical stress treatment. Cell viability was measured by MTT assay. B Gene expression of Piezo1 was measured by real-time PCR. C , D Western blot analysis of Piezo1’s protein level. Total β-actin served as loading controls. E , F Immunofluorescence staining analysis of Piezo1 expression. Relative fluorescent levels of Piezo1 were measured by Image J software. G Concentrations of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in supernatants were measured by ELISA. H Mitochondrial membrane potential was measured by JC-1 probe and flow cytometer. I OCR of cells were measured by Seahorse XFe96 Extracellular Flux Analyzer at basal conditions and with serial administration of oligomycin, FCCP and rotenone. J – L Gene and protein expressions of P53 and P16. Total β-actin served as loading controls. * p < 0.05. All experiments were repeated at least three times

Article Snippet: After deparaffinization, rehydration, antigen retrieval, and blocking, the slides were incubated with primary antibodies against Piezo1 (No. NBP1-78537, Novus Biologicals, USA, 1:100 dilution) and type II Collagen (No. NBP1-77795, Novus Biologicals, USA, 1:200 dilution) on a humidified box at 4 °C overnight.

Techniques: Expressing, Cell Function Assay, MTT Assay, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Software, Enzyme-linked Immunosorbent Assay, Membrane, Flow Cytometry

Journal: Arthritis Research & Therapy

Article Title: Excessive mechanical stress-induced intervertebral disc degeneration is related to Piezo1 overexpression triggering the imbalance of autophagy/apoptosis in human nucleus pulpous

doi: 10.1186/s13075-022-02804-y

Figure Lengend Snippet: Autophagy and apoptosis related protein expressions in human nucleus pulposus cells after mechanical stress treatment. A Concentrations of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in supernatants after mechanical stress treatment with/without administration of Piezo1 inhibitor GsMTx4. B Mitochondrial membrane potential was measured after mechanical stress treatment with/without GsMTx4. C – E Western blot analysis of protein levels of Col II, Aggrecan, LC3 II, LC3 I, Beclin-1, P62. Total β-actin served as loading controls. F , G Immunofluorescence staining analysis of LC3 expression after mechanical stress treatment with or without GsMTx4, relative fluorescent levels of LC3 were measured by Image J software. H , I Western blot analysis of Bax and Bcl-2’s protein levels. Total β-actin served as loading controls. J Cell apoptosis was assessed by flow cytometer with Annexin V-FITC/PI apoptosis detection kit. * p < 0.05. Data were obtained from three independent experiments

Article Snippet: After deparaffinization, rehydration, antigen retrieval, and blocking, the slides were incubated with primary antibodies against Piezo1 (No. NBP1-78537, Novus Biologicals, USA, 1:100 dilution) and type II Collagen (No. NBP1-77795, Novus Biologicals, USA, 1:200 dilution) on a humidified box at 4 °C overnight.

Techniques: Membrane, Western Blot, Immunofluorescence, Staining, Expressing, Software, Flow Cytometry

Journal: Arthritis Research & Therapy

Article Title: Excessive mechanical stress-induced intervertebral disc degeneration is related to Piezo1 overexpression triggering the imbalance of autophagy/apoptosis in human nucleus pulpous

doi: 10.1186/s13075-022-02804-y

Figure Lengend Snippet: Excessive mechanical stress stimulates mechanosensitive channel protein Piezo1 overexpression in human nucleus pulposus cells, which aggravates mitochondrial damage and inflammatory response in NP cells and further triggers the reduction of autophagy and the intensification of apoptosis. The imbalance between autophagy and apoptosis may result in cell senescence and dysfunction, weaken the synthesis of collagen II and Aggrecan in NP cells, and consequently precipitate cell death and exacerbate disc degeneration

Article Snippet: After deparaffinization, rehydration, antigen retrieval, and blocking, the slides were incubated with primary antibodies against Piezo1 (No. NBP1-78537, Novus Biologicals, USA, 1:100 dilution) and type II Collagen (No. NBP1-77795, Novus Biologicals, USA, 1:200 dilution) on a humidified box at 4 °C overnight.

Techniques: Over Expression