Fig. 5: Cytoplasmic MYC degradation is associated with changes in the interactome of hyperacetylated tubulin.

A Interaction of MYC and HDAC6 detected by IP. Ramos cells were treated for 24 h with 4 µM M-100 or left untreated. IPs with unspecific IgG were used as control. B Cytoplasmic and nuclear fractions were prepared from Ramos cells. HDAC1 was used as a nuclear marker and tubulin as a cytoplasmic marker. C NIH-3T3 cells overexpressing MYC-GFP were challenged for 1 h with inhibitors of nuclear import (Importazole, IMP; 40 µM), nuclear export (Leptomycin B, LMB; 20 ng/ml), or solvent. Next, cells were treated for 90 min with CHX (50 µg/ml) before flow cytometry. Living cells were gated using FSC/SSC and normalized median fluorescence intensity (MFI) of MYC-GFP was calculated. Data represent mean + SEM. Unpaired Student’s t-test, two-tailed. D PLA was performed to detect endogenous co-localization of MYC and acetylated tubulin (ac-tub) in Ramos cells. Cells were treated with 4 µM M-100 for 24 h. Staining with unspecific IgG was used as a control. DAPI was used to stain nuclei. Scale bars indicate 20 µm, magnification 60x. PLA foci were counted and compared. Boxplots depict medium and min to max. One-Way ANOVA (Tukey’s posthoc). E Global interactome analysis was carried out of immunoprecipitated ac-tub via mass spectrometry. MV4-11 cells were treated for 24 h with 0.5 µM M-100. Shown are counts of proteins with new or increased (>2-fold) binding to ac-tub, or loss of binding after treatment compared to control and IgG binding. Uniprot (UP) keyword annotation was performed with DAVID using all proteins that bound to ac-tub after M-100 treatment. Adjusted (adj.) P-values are given. Ubl - Ubiquitin-like. F All proteins belonging to the keyword “chaperone” are depicted with their corresponding log2 fold change (FC). DNAJ proteins are marked in green. Data in A and C are representative of n = 3 independent experiments. Data in B and D are representative of n = 2 independent experiments.