Fig. 1

Inactivation of SPOP/CK1 signaling destabilizes ACE2 proteins to alleviate SARS-CoV-2 S protein conditioned pseudoviral infection. a Immunoblot (IB) analysis of whole cell lysates (WCL) derived from indicated cell lines. b A cartoon illustration to demonstrate that the putative SPOP degron is located within the first five amino acids in the ACE2 signal peptide motif. c IB analyses of WCL derived from indicated cells depleted of endogenous SPOP by lenti-viral shRNAs. Cells were selected with 1 μg/ml puromycin for 72 h to eliminate non-infected cells before cell collection. d IB analyses of WCL and Ni-NTA pulldowns derived from HEK293T cells transfected with indicated DNA constructs. 10 μM MG132 was added to cell culture 10 h prior to cell collection. e Top, an illustration of the ACE2 mutation and deletion generated in this panel. Bottom, IB analyses of WCL and Flag-IPs derived from HEK293T cells transfected with indicated DNA constructs. f IB analyses of WCL derived from UMRC6, RCC4, and UMRC2 cells depleted of indicated endogenous CK1 isoforms by lenti-viral shRNAs. Cells were selected with 1 μg/ml puromycin for 72 h to eliminate non-infected cells before cell collection. g IB analyses of WCL and Flag-IPs derived from HEK293T cells transfected with pLenti-ACE2-HA and Flag-SPOP. Indicated doses of D4476 and 10 μM MG132 was added to cell culture 10 h prior to cell collection. h IB analyses of WCL and Ni-NTA pulldowns derived from HEK293T cells transfected with indicated DNA constructs. 20 or 40 μM D4476 with 10 μM MG132 were added to cell culture 10 h prior to cell collection. i IB analyses of WCL and Ni-NTA pulldowns derived from HEK293T cells transfected with indicated DNA constructs. 10 μM MG132 was added to cell culture 10 h prior to cell collection. j IB analysis of WCL derived from HEK293T cells transfected with indicated CMV-GST-ACE2 constructs. 3 or 30 μM lenalidomide was added to cell culture 10 h prior to cell collection. k IB analyses of WCL and Flag-IPs derived from HEK293T cells transfected with CMV-GST-ACE2 and Flag-SPOP. Indicated doses of lenalidomide was added to cell culture 10 h prior to cell collection. l IB analyses of WCL and Ni-NTA pulldowns derived from HEK293T cells transfected with indicated DNA constructs. 40 μM lenalidomide and 10 μM MG132 were added to cell culture 10 h prior to cell collection. m, n IB analyses of WCL derived from UMRC2 cells treated with indicated doses of CK1 inhibitor D4476 (m) or epiblastin A (n) for 10 h. o–q IB analyses of WCL derived from UMRC2 (o), UMRC6 (p), and Calu-3 (q) cells treated with indicated doses of CK1α PROTAC lenalidomide for 10 h. r A cartoon illustration of a proposed model that lenalidomide targets CK1 for CRBN-mediated degradation, alleviating SPOP binding and protection on ACE2 proteins, therefore leading to reduced ACE2 protein abundance. s IB analyses of WCL derived from endogenous ACE2-depleted UMRC2 cells infected with indicated pLenti-ACE2-HA viruses. t Representative immunofluorescent images using WT- and 3A-ACE2 UMRC2 cells obtained from (s) immune-stained with an HA antibody to indicate ACE2 signals. u Pretreating UMRC2 cells with 80 μM lenalidomide for 24 h reduced infection by GFP expressing SARS-CoV-2 S protein conditioned pseudoviruses in vitro. GFP signals were measured 24 h post-infection. Please refer to “Methods” section for details. Error bars were calculated as mean +/− SD, n = 3. *p < 0.05 (one-way ANOVA test). v IB analyses of WCL derived from UMRC2 cells with indicated treatments. Where indicated, 40 μM lenalidomide was added to cell culture 7 h prior to cell collection. w GFP expressing SARS-CoV-2 S protein conditioned pseudovirus infection and lenalidomide treatment do not significantly affect cell proliferation in vitro determined by CellTiter-Glo assays. Error bars were calculated as mean +/− SD, n = 3. *p < 0.05 (one-way ANOVA test). x Representative immunofluorescent images indicating that lenalidomide pre-treatment reduces infection of UMRC2 cells by GFP expressing SARS-CoV-2 S protein conditioned pseudoviruses in vitro. The bar indicates 50 μm. y Pre-treatment of UMRC2 cells with lenalidomide reduces infection by home-made luciferase-expressing SARS-CoV-2 S protein conditioned pseudoviruses in vitro. UMRC2 cells were treated with 40 μM lenalidomide for 7 h before pseudoviral infection in all groups. Group 1, UMRC2 cells were infected with 500 μL home-made pseudoviruses then treated with 40 μM lenalidomide immediately after spinoculation. Group 2, UMRC2 cells were infected with 500 μL home-made pseudoviruses then treated with 40 μM lenalidomide immediately after spinoculation, followed by refreshing culture with 40 μM lenalidomide 24 h post-spinoculation. Group 3, UMRC2 cells were infected with 500 μL home-made pseudoviruses then treated with 40 μM lenalidomide immediately after spinoculation, followed by refreshing culture with 40 μM lenalidomide 24 and 48 h post-spinoculation, respectively. Viral infection was monitored by RLU (relative light unit) generated in luciferase activity assays. Error bars were calculated as mean +/− SD, n = 2. *p < 0.05 (one-way ANOVA test). z IB analyses of WCL derived from indicated ACE2 expressing UMRC2 cells treated with indicated doses of lenalidomide for 20 h prior to cell collection