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

Figure 4

Ca²⁺ accumulation leads to mitochondria-related programmed cell death via the PARP/AIF pathway. (a) Representative flow cytometry images of PI uptake analysis for NP cells that were treated with 5 μM RR for 1 h and then exposed to compression for 36 h. (b) Quantitative analysis of flow cytometry for cellular PI uptake. NP cells were treated as in (a). The values are expressed as the from three biological replicates ( vs. control, ^## vs. compression alone, ANOVA/LSD). (c) Representative western blot graphs of the levels of HMGB1 content in the media. NP cells were treated as in (a). (d) Histogram for statistical analysis of the LDH leakage in compression-treated NP cells. NP cells were treated as in (a). The values are expressed as the from three biological replicates ( vs. control, ^### vs. compression alone, ANOVA/LSD). (e, f) Typical fluorescence photomicrographs and quantitative analysis of mitochondrial membrane potential performed by a JC-1 probe. NP cells were treated as in (a). The values are expressed as the from three biological replicates ( vs. control, ^### vs. compression alone, ANOVA/LSD). (g) Quantitative analysis of ATP production in NP cells. NP cells were treated as in (a). The values are expressed as the from three biological replicates ( vs. control, ^## vs. compression alone, ANOVA/LSD). (h) Representative western blot graphs of PARP and C-PARP determined by WB. NP cells were treated as in (a). (i) Representative western blot graphs of mitochondrial and intranuclear AIF protein levels determined by WB. NP cells were treated as in (a). (j) Representative fluorescence images of the AIF staining in NP cells. NP cells were stained for nuclei by DAPI (blue) and for AIF by anti-AIF antibody (red). NP cells were treated as in (a).

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