Methods (MDC staining): Arabidopsis cells were stained with a 0.05 mM final concentration of MDC in PBS for 10 min. When staining protoplasts, PBS was supplemented with 0.4 M mannitol. Cells were washed with PBS two times to remove excess MDC. Fluorescence of suspension cells was measured using a Hitachi F-2000 fluorescence spectrophotometer with anexcitation wavelength of 335 nm and an emission wavelength of 508 nm. The intensity of MDC staining was expressed in relative fluorescence per gram of fresh weight, as a percentage of the 0-h control.

Results: MDC stains autophagosomes in Arabidopsis, as it does in mammaliancells.

Methods (MDC staining): Cells were seeded in 24-well culture plates and treated with compression for 0, 12, 24, 36 and 48 h. The autophagy inhibitor 3-MA was applied to assay the effect of compression on IVD cell autophagy. At each time point following treatment, cells were rinsed in PBS (three times for 5 min), incubated with a 0.05 mM solution of MDC dye (diluted from the 0.5 M stock solution in DMSO) at 37C for 15 min, and then washed three times in PBS.