Fig. 7.

Implication of lysosomal cathepsins in bupivacaine-induced cell death. (A) Typical confocal images of cathepsin D immunofluorescence staining in IVD cells after exposure to saline or bupivacaine (Magnification ×800, scale bars = 20 µm). (B) Representative graphs obtained from flow cytometry analysis after Annexin V/PI staining. Before exposure to bupivacaine, the IVD cells were pretreated with or without the cathepsin B inhibitor CA074-Me (20 μM) or the cathepsin D inhibitor pepstatin A (20 μM) for 60 min. After bupivacaine treatment, cell death was assessed by flow cytometry. (C) Histogram analysis showing the percentage of PI-positive cells. Data are presented as the means ± SD of three independent experiments (^*P < 0.05, CA-treated or PstA-treated vs. corresponding untreated cells). AF, annulus fibrosus; NP, nucleus pulposus; B, bupivacaine; CA, CA074-Me; PstA, pepstatin A.