Fig. 5: SKP2 inhibitors enhance BECN1 protein stability and autophagy.

a, b Evaluation of small compounds inhibiting SKP2. HEK293 cells were exposed to known SKP2 inhibitors for 24 h, followed by additional exposure to cycloheximide (CHX, 30 µg mL⁻¹) for the times as indicated or vehicle (a). b HEK293 cells were transfected with Halo-tagged BECN1 expressing plasmid and exposed to known SKP2 inhibitors and halogenated dye (100 nM, R110Direct) overnight. Cells were transferred to medium without dye and harvested after the indicated times. Labelled BECN1 was determined and quantified. c HEK293 cells were transfected with control vector (−) or plasmids expressing BECN1-Flag, HA-ubiquitin and myc-SKP2. Cells were exposed to known SKP2 inhibitors (C1, 3 µM; SMER3, 5 µM; SMIP004, 10 µM) for 24 h, BECN1 was precipitated from cell extracts and its ubiquitination and levels were determined by western blotting. d SKP2 inhibitors impact long-lived proteins. HEK293 cells were metabolically labelled with [¹⁴C]-valine overnight, exposed to the indicated drugs or starvation (HBSS = Hank’s balanced salt solution) and the degradation of proteins was determined 4 h after withdrawal of [¹⁴C]-valine completed media. The autophagy inhibitor 3-MA (10 mM) was used as control. e, f Assessment of autophagy markers upon exposure to SKP2 inhibitors. VeroB4 cells were exposed to the indicated drugs or vehicle and the levels of P62 and LC3B-II/I were determined by western blotting (e). The most efficient drug, SMIP004, was analyzed in the flux assay with bafilomycin A1 (BafA1; f). In all panels, error bars denote the standard error of the mean, derived from n = 3 biologically independent experiments. WCE = whole cell extract. *^,$p < 0.05, **^,$$p < 0.01, ***^,$$$p < 0.001 (two-way ANOVAs, details in Supplementary Table 1). In d, *labels refer to the effects of 3-MA, ^$labels to the drug effects in comparison to vehicle. Source data and blot collections are provided as a Source Data file.