ti2 inverted fluorescence microscope  (Nikon)

Journal: Journal of Orthopaedic Translation

Article Title: Oxidative stress activates YAP/TEAD1/NCOA4 axis to promote ferroptosis of endplate chondrocytes and aggravate intervertebral disc degeneration

doi: 10.1016/j.jot.2025.07.001

Figure Lengend Snippet: YAP activation in IDD: Correlation with oxidative stress and ferroptosis ( A, B ) Immunohistochemistry for YAP in cartilage endplate from each group. Scale bar = 100 μm. The ratio of positive cells for YAP was quantified under a microscope at 400 × magnification. ( C, D ) endplate chondrocytes were isolated and treated with increasing concentrations (0,10,50,100,200 μM) of TBHP for 6 h, and Western blot was conducted to examine the protein levels of YAP and pYAP. The band densities of YAP and pYAP were quantified and normalized to the control group to calculate the ratio between the active and inactive forms (YAP/pYAP). ( E, F ) endplate chondrocytes were pre-treated with TBHP (100 μM) for 6 h and immunofluorescence staining was performed to examine the expression of YAP (red). Scale bar = 50 μm. Quantitative analysis of red fluorescence intensity was performed. Data are presented as mean ± SD from 3 or 5 independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Article Snippet: In summary, following a 30-min incubation period with the JC-1 staining solution at room temperature, endplate chondrocytes were rinsed three times with pre-cooled JC-1 washing buffer and subsequently observed under an inverted fluorescence microscope (Axio Observer A1; Carl Zeiss, Germany).

Techniques: Activation Assay, Immunohistochemistry, Microscopy, Isolation, Western Blot, Control, Immunofluorescence, Staining, Expressing, Fluorescence

Journal: Journal of Orthopaedic Translation

Article Title: Oxidative stress activates YAP/TEAD1/NCOA4 axis to promote ferroptosis of endplate chondrocytes and aggravate intervertebral disc degeneration

doi: 10.1016/j.jot.2025.07.001

Figure Lengend Snippet: Targeting the regulation of NCOA4-mediated ferritinophagy through the inhibition of the YAP/TEAD1 axis to ameliorate the degeneration of endplate chondrocytes ( A ) Endplate chondrocytes were isolated and treated with 100 μM TBHP with increasing concentrations (0,0.01,0.1,0.25,0.5,1and 2 μM) of VP for 24 h, CCK assay was conducted to evaluate the cell viability. ( B, C ) Endplate chondrocytes were treated with or without VP(0.25 μM) following TBHP (100 μM) induction for 6 h. Western blot was conducted to examine the protein levels of YAP, TEAD1, GPX4, NCOA4 and FTH. The band densities were quantified and normalized. ( D, E ) Endplate chondrocytes were treated with or without VP(0.25 μM) following TBHP (100 μM) induction for 6 h. Immunofluorescence staining was performed to examine the expression of NCOA4 (red). Scale bar = 50 μm. Quantitative analysis of red fluorescence intensity was performed. ( F, G ) Immunohistochemistry for TEAD1 and NCOA4 in cartilage endplate from each group. Scale bar = 100 μm. The ratio of positive cells for TEAD1 and NCOA4 was quantified under a microscope at 400 × magnification. ( H ) NCOA4 was overexpressed and transfection efficiency was assessed using Western blot to determine NCOA4 expression. ( I-L ) Endplate chondrocytes were divided into OE-NC (overexpression-NC plasmid) + TBHP group, OE-NC + TBHP + VP group and TBHP + VP + OE-NCOA4 (overexpression-Ncoa4 plasmid) group according to the treatment. Western blot was conducted to examine the protein levels of GPX4, NCOA4, FTH, COL2, MMP3, CTGF, COL10 and RUNX2. The band densities were quantified and normalized. ( M ) Immunohistochemistry for COL2 in cartilage endplate from each group. Scale bar = 100 μm. The ratio of positive cells for COL2 was quantified under a microscope at 400 × magnification. Data are presented as mean ± SD from 3 or 5 independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Article Snippet: In summary, following a 30-min incubation period with the JC-1 staining solution at room temperature, endplate chondrocytes were rinsed three times with pre-cooled JC-1 washing buffer and subsequently observed under an inverted fluorescence microscope (Axio Observer A1; Carl Zeiss, Germany).

Techniques: Inhibition, Isolation, Western Blot, Immunofluorescence, Staining, Expressing, Fluorescence, Immunohistochemistry, Microscopy, Transfection, Over Expression, Plasmid Preparation