SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection

Gassen, Nils C.; Niemeyer, Daniela; Muth, Doreen; Corman, Victor M.; Martinelli, Silvia; Gassen, Alwine; Hafner, Kathrin; Papies, Jan; Mösbauer, Kirstin; Zellner, Andreas; Zannas, Anthony S.; Herrmann, Alexander; Holsboer, Florian; Brack-Werner, Ruth; Boshart, Michael; Müller-Myhsok, Bertram; Drosten, Christian; Müller, Marcel A.; Rein, Theo

Supplementary Figure 1 . Supplements to main Figures 1-3 . a Quantification referring to main Figure 1b . b BECN1 is subject to proteasomal degradation. HEK293 cells were transfected with Ubiquitin-HA expressing plasmid and treated with the proteasome inhibitor MG132 (10 µM, 2 h) in combination with NH 4 Cl (10 mM) as indicated. BECN1 was immunoprecipitated from whole cell extracts and probed for ubiquitination by western blotting 1 . c Quantification referring to main Figure 2g . d Quantification referring to main Figure 2h . e Lysine 402 is situated at an accessible site on the surface of BECN1. Depicted is the crystal structure of the evolutionary conserved domain of BECN1 (dbd # 4DDP_A) 2 with K402 highlighted in yellow. f Toxicity assays evaluating the PHLPP inhibitor (PHLPPi; NSC117079). VeroB4 cells were treated with the inhibitor (used in Figure 4d -h) at increasing concentrations as indicated for 1 h and cell viability was determined by the LDH (lactate dehydrogenase) and the MTT (tetrazole 3-(4,5dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide) assay as described previously 3 . g Western blot and quantification of P62 upon downregulation of SKP2 by siRNA in HEK293 cells. In all panels, error bars denote the standard error of the mean, derived from n=3 (a-c,g) or n=6 (d) biologically independent experiments. * p<0.05, ** p<0.01, *** p<0.001 (b, 1 way ANOVA; a,c,d,g, t-tests; details in Supplementary Tables 1 and 2) . Source data are provided as a Source Data file. VeroB4 cells were treated with the SKP2 inhibitors C1, SMER3 and SMIP004 at increasing concentrations as indicated for 24 h and cell viability was determined by the MTT (tetrazole 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide) assay as described previously 3 . Based on these results, the concentrations used in the experiments of the main figures were chosen (i.e. at least 85% cell viability). b,c Titration of the BafA1 effect on LC3B lipidation in VeroB4 cells as required for the flux assays 1 . VeroB4 cells were exposed to increasing concentrations of BafA1 for 2 h before cells were lysed. The ratios of LC3B-II/I were determined by western blotting (a representative blot is shown). The graph represents the average levels + SEM of three independent experiments. Based on these data, 100 nM BafA1 was chosen for the experiments in all figures assessing autophagic flux as the concentration achieving complete block of autophagosome-lysosome fusion 1 . d HBSS control in VeroB4 cells to demonstrate the induction of autophagic flux. Representative Western blots are displayed. e-g VeroB4 cells were treated with the indicated inhibitors (C1 (3.3 µM), SMIP004 (10 µM), SMER3 (5 µM)) and the indicated protein levels were determined (mean + SEM of three independent experiments; quantification to the western blots in Figure 5e ). h,i VeroB4 cells were exposed to SMIP004 for 24h, cross-linked with disuccinimidyl suberate (DSS, 75 µM) for 30 min and harvested. ATG14 homo-oligomerization was examined after western blotting (ProteinSimple). Quantification in (i). In all panels, error bars denote the standard error of the mean, derived from n=6 (a) or n=3 (c-g,i) biologically independent experiments. * p<0.05, ** p<0.01, *** p<0.001 (1 way ANOVA for b,e-g, 2 way ANOVA or d, and t-test for i, details in Supplementary Tables 1 and 2) . Source data are provided as a Source Data file.