Extended Data Fig. 4: CHX is reducing translation in nematodes.
a) Output of the Dmin function of the Mfuzz R package. Soft clustering for cluster numbers ranging from 2-12 (x-axis) were calculated with Dmin. For each cluster number, Dmin calculates the distance between the centroids of the clusters (centroid distance) and reports the minimum centroid distance across 3 repetitions (y-axis). The optimal cluster number is estimated from the “elbow” of the plot (indicated by an orange arrow on the dashed line), that is the cluster number which shows a sharp decline in the minimum centroid distance (cluster Number=4). b) SUnSET assay to test translation efficacy upon cycloheximide treatment. Left side: Westernblot detecting puromycin labelled proteins, ponceau staining as loading control, left side: densitometric quantification normalized to total protein according to ponceau signal. Four independent cohorts of each ≈ 35 nematodes tested. One-tailed t-test to test for significance. Lysed nematodes were day 1 of adulthood, 2 mM CHX treatment since L4 stage and while puromycin labelling. c) Fluorescence quantification to assess GFP synthesis in selected neurons as proxy for translation. Three independent cohorts, total 28 – 52 nematodes analysed per strain and condition. One-way ANOVA + Tukey post hoc test. d) Schematic depiction of experimental procedure to test the effect of either short-term or chronic CHX treatment on the neuron integrity. e) Fraction plots of neuronal integrity of ASK, ASJ, ASE, and OLL neurons treated with 2 mM CHX for either 24 h or 96 h starting from L4 stage on. Three cohorts of 4 – 20 nematodes expressing GFP in either ASK, ASJ, ASE, or OLL neurons have been recorded, neurodegeneration was assessed, and neurons categorized as ‘healthy’ (blue), ‘mildly damaged’ (orange), and ‘severely damaged’ (red). Ordinal regression (cumulative link model) has been used to test for significant differences between CHX and solvent control treatment. To test for significant differences a cumulative link model was built, pairwise comparison was corrected according to Bonferroni.
