Fig. 4

Proteasome bulk activity is quickly stimulated by high glucose delivery. A Whole cell extracts of rMC1 cells stimulated with 25 mmol/L glucose and with low glucose or mannitol for 10, 20 and 40 min were probed with antibodies anti-ubiquitin, -p21 and -p53; GAPDH was used as loading control by Wb analysis. Histograms show the densitometric analysis of the immunoreactive bands; data are the mean ± SD of three independent experiments; a representative blot is shown. Statistics were calculated for high glucose vs low glucose treated cells; one-way ANOVA followed by Tukey’s post-hoc test: *p < 0.05, **p < 0.01. B rMC1 cells were pre-treated with 500 nM epoxomicin or vehicle (DMSO) for 1 h. Thereafter, the compound was washed out before addition of high glucose or controls. After 40 min of incubation, whole cell lysates were prepared, analysed by Wb and probed with the anti-ubiquitin antibody. GAPDH was used as loading control; raw normalized values of band intensity are reported below the upper panel. C Crude cell extracts were isolated from rMC1 cells challenged with high glucose as indicated in A and bulk proteasome activity assayed on Suc-LLVY-amc, a fluorogenic peptide specific for the chymotrypsin-like activity. Values reported in the graph are the relative ratio of the slope calculated for high-glucose treated cells vs low-glucose or mannitol-treated cells at each time-point. Peptide cleavage was monitored until linearity was observed (~ 40 min). The time (x) scale refers to the timing after glucose stimulation; slopes have been determined by subtracting that obtained in the presence of 500 nM epoxomicin (added to the crude cell extract) for each experimental condition. Non-specific cleavage of the fluorogenic probe in the presence of epoxomicin was negligible. One-way ANOVA followed by Tukey’s post-hoc test (n = 3): *p < 0.05; **p < 0.01. D Crude cell extracts of cells treated with low glucose and high glucose were separated by native-gel electrophoresis. The activity of proteasome particles was visualized in-gel upon delivery of 75 µM Suc-LLVY-amc. The identity of capped species was determined by Wb analysis, probing the filters with an anti-Rpt5 antibody. An aliquot of these extracts was further analysed by denaturing and reducing Wb and probed with anti-α4, -Rpt5 and -Rpt6 antibodies; a representative experiment of three independent replicates is reported. E Living rMC1 cells were pre-stimulated with 500 nM epoxomicin or DMSO for 1 h. Thereafter, the compounds were washed out and the stimuli (i.e., low glucose, high glucose and mannitol) delivered. After additional 5 min of incubation the TED fluorogenic peptide (17 µM) was added to the culture medium and fluorescence release monitored over 20 min of high glucose stimulation. Arrows indicate the precise timing of stimuli administration. Data are reported as relative comparison between Relative Fluorescence Unit (RFU) obtained at each time-point. In accordance with previous results, preliminary data indicated that TED half-life is quite short (around 30 min. and that non-specific cleavage of the peptide was almost null, since epoxomicin treated cells did not release appreciable fluorescence (data not shown) [32]. One-way ANOVA followed by Tukey’s post-hoc test (n = 3): **p < 0.01