Fig. 1

Increased tolerance to neuronal proteasome inhibition in the presence of astrocytes. a LUHMES cells (d6) were treated with the indicated concentrations of MPP⁺ or rotenone for 24 h. Proteasome activity was assessed fluorometrically. b LUHMES cells (d6) were treated with MPP⁺ [5 µM] either in mono- or in co-culture with astrocytes for the indicated time periods. Viability was assessed by measuring resazurin reduction at the indicated time points. c LUHMES astrocyte co-cultures were treated with MPP⁺ [5 µM] or rotenone [1 µM] for 24 h. Proteasome activity was assessed fluorometrically. d LUHMES cells in mono- and co-culture with astrocytes were differentiated according to the depicted differentiation scheme. LUHMES cells were replated at d-1, and differentiation started by replacing the proliferation medium (PM) with differentiation medium (DM) containing tetracycline, cAMP and GDNF on d0. After 2 days (d2), LUHMES cells were re-plated either as mono-cultures or seeded on top of pre-differentiated astrocytes (mAGES). The medium was exchanged on d4 and 'mature cells' were ready on d6 for toxicant exposure. e LUHMES cells (d6) mono- or co-cultures were exposed to the indicated concentrations of MG-132 for 24 h. Differential toxicity was assessed by immunocytochemistry staining with antibodies against β-III tubulin and GFAP. Nuclei were counterstained with H-33342. f Toxicity of MG-132 on LUHMES mono- and co-cultures was assessed by measuring the neurite integrity after cells were exposed for 24 h to MG-132 at the indicated concentrations. g Proteasomal inhibition by MG-132 in LUHMES mono- and co-cultures was assessed by measuring proteasome activity fluorometrically. For A-F, differences were tested for significance by one-way ANOVA, followed by Dunnett’s post hoc test, n.s.: non-significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001 for comparison of treatments to the respective untreated controls. Data are means ± SD of three independent experiments