Fig. 2: Disruption of the endoplasmic reticulum (ER)-mitochondria interaction network in human intervertebral disc degeneration (IVDD).

a Schematic diagram of RNA-seq. Created in BioRender. Chen, P. (2026) https://BioRender.com/6rte857. b Principal component analysis (PCA) of genes. c Heatmap showing differentially expressed genes in different groups; three biological replicates are shown. d Volcano plots of differentially expressed genes. P values were calculated using DESeq2 analysis with the Wald test. e Gene set enrichment analysis (GSEA) results of different groups. f Simultaneous imaging (left) and quantification (right) of cytosolic and mitochondrial Ca²⁺ using Fluo-4/AM and Rhod-2/AM (n = 3). g Immunofluorescence staining (left) and quantification (right) of CHOP and ATF4 protein levels (n = 3). h Representative confocal image of human NPCs stained for calnexin (green, ER) and TOMM20 (red, mitochondria). Merged panel images were processed using Fiji/ImageJ (white dots indicate colocalized pixels). i Mander’s coefficients (M1) of (h). n = 3 biological replicates, 20 images were acquired and analyzed from each group. j Transmission electron microscopy (TEM) images of mitochondrial and ER morphology. The ER and mitochondria were graphically reconstructed: mitochondria (red); ER (green). k Quantification of the mitochondrial–ER contact length in (j). l Mitochondrial count per field from TEM in (j). m Quantification of ER lengths per field from (j). Measurements in (k–m) are from n = 3 biological replicates, with the normal group = 50 cells from 65 fields and the degeneration group = 50 cells from 63 fields. OMM: Outer mitochondrial membrane. n Calcein fluorescence (left) and quantification (right) of human NPCs (n = 3). o Reactive oxygen species (ROS) staining (left) and quantification (right) of human NPCs (n = 3). p JC-1 staining (left) and quantification (right) of human NPCs (n = 3). All quantitative data are the mean ± s.d. n represents the number of biologically independent samples. P values were calculated using two-tailed Student’s t test (f, g, i, and k–p). Scale bars: 50 µm (f–h, n–p); 1 µm (j). Source data are provided as a Source Data file.