Fig 2: SKP2 inhibition reduces replication of MERS-CoV and its effects on autophagy.a The SKP2 inhibitor (SKP2i) efficiently inhibits MERS-CoV replication. VeroB4 cells were infected with MERS-CoV (MOI = 0.001) and treated with SKP2i (10 µM) or DMSO (Co = control). MERS-CoV GE were determined by real-time RT-PCR at 24 and 48 h p.i., data are presented as fold difference (black bars, SKP2i condition) in comparison to control (gray bars, Co = DMSO condition). Raw data presenting GE ml^-1 is shown in Fig. S5A. b, c The SKP2-inhibitor (SKP2i) restores autophagic flux in MERS-CoV-infected cells. VeroB4 cells were transfected with mRFP-EGFP-tagged LC3B, infected with MERS-CoV (MOI = 0.001) and treated with SKP2i (10 µM) or vehicle for 24 h. b Representative images (scale bar 25 µm). c The numbers of vesicles with both green and red fluorescence (autophagosomes, AP) and with red fluorescence only (autolysosomes, AL) were counted 24 h p.i.. d–f SKP2i enhances protein interactions indicative of autolysosome formation in MERS-CoV-infected cells. VeroB4 cells were infected with MERS-CoV (MOI = 0.001), treated with SKP2i for 48 h, cross-linked with disuccinimidyl suberate (DSS, 75 µM) 48 h p.i. for 30 min and harvested. ATG14 homo-oligomerization was assessed after western blotting (d) and quantified (e). f The SNARE complex protein STX17 was immunoprecipitated and the eluate was probed for interacting VAMP8 and SNAP29 by western blotting and quantified as in Fig. 6h. In all panels, error bars denote the standard error of the mean, derived from n = 3 (a, e) or n = 4 (f) biologically independent experiments, and n = 13 (c, vehicle, non-infected) or n = 12 (all other conditions in c) different cells. *p < 0.05, **p < 0.01, ***,###p < 0.001 (c, *,***refer to the statistical difference between the numbers autolysosomes, ###to the difference between the total numbers of fluorescing vesicles). Two-way ANOVA was performed in c, t-tests in a, e, f, details in Supplementary Tables 1, 2. Source data and blot collections are provided as a Source Data file.

Fig 3: VAMP2 is involved in EA-regulated FPN translocation.(A) mRNA expression levels of Vamp (Vamp2, Vamp8 and Vamp8), SNAP23 and syntaxin 4 in primary HSCs from C57BL/6 mice and LX-2 cells. n = 4. (B) Protein expression of VAMP2, VAMP3, VAMP8, syntaxin 4, and SNAP23 in primary HSCs and LX-2 cells treated with TGF-β1 and/or EA. n = 5. (C) VAMP2 expression in primary HSCs and LX2 cells pretreated with MG-132 (10 μM) or CQ (1 μM) for 30 min followed by TGF-β1 and/or EA treatment. n = 5. (D) Following TGF-β1 treatment, VAMP2 expression was analyzed in primary HSCs and LX2 cells pretreated with CHX (100 μg/mL) for the indicated durations. n = 5. (E) Immunoblotting analysis of syntaxin 4 and SNAP23 in VAMP2 immunoprecipitates from primary HSCs and LX2 cells treated with TGF-β1 and/or EA. n = 4. (F) Primary HSCs and LX-2 cells were infected with different concentrations of AdshVAMP2, AdVAMP2 or control adenovirus (AdLacz) for 24 h. VAMP2 protein expression was examined. n = 5. (G) After HSCs were infected with AdshVAMP2 or AdVAMP2 followed by TGF-β1 treatment, the cells were incubated in EA for 24 h, and then the distribution of FPN and (H) FTH1 expression in those cells was examined by western blotting. n = 5. Data were normalized to GAPDH and are expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 between the indicated groups.