Reactive Oxygen Species Regulate Endoplasmic Reticulum Stress and ER-Mitochondrial Ca²⁺ Crosstalk to Promote Programmed Necrosis of Rat Nucleus Pulposus Cells under Compression

Figure 3

Compression-induced Ca²⁺ communication between the ER and the mitochondria relies on the IP₃R–GRP75–VDAC1 complex. (a) Representative fluorescent images of the colocalization of IP₃R1 and VDAC1 in NP cells treated with compression for 0, 12, 24, and 36 h. In the merged images, overlap (yellow), representing the colocalization of IP₃R1 and VDAC1, was obviously increased after 36 h mechanical loading. (b, c) Representative western blot graphs of coimmunoprecipitations of IP₃R1, GRP75, and VDAC1 in NP cells treated with compression for 36 h. (d) The typical western blot bands of VDAC1 and GRP75 in NP cells transfected with negative control siRNA (NC), VDAC1-siRNA, and GRP75-siRNA. (e, f) Representative histograms and statistical analysis of [Ca²⁺]_m detected by flow cytometry in compression-treated NP cells with Rhod-2 AM. NP cells were pretreated with VDAC1-siRNA or GRP75-siRNA and then exposed to 1 MPa compression for 36 h. The values are expressed as the from three biological replicates ( vs. control, ANOVA/LSD). (g, h) Representative dot plots and quantitative analysis of PI uptake in NP cells. NP cells were pretreated with 2.5 μM XeC, VDAC1-siRNA, or GRP75-siRNA and then exposed to 1 MPa compression for 36 h. The values are expressed as the from three biological replicates ( vs. compression alone, ANOVA/LSD).

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