Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Schematics of the cell-cell fusion model used to quantify spike-mediated syncytium formation upon treatment with cell culture supernatants from Toll-like receptor (TLR) ligands-stimulated innate immune cells. Cells co-expressing SARS-CoV-2 spike and Cre were co-cultured with ACE2 and Stop-luc co-expressing HEK293T cells for 16 hr, before cell lysates were collected for bioluminescence assay and immunoblotting. Cells co-expressing SARS-CoV-2 spike and ZsGreen were co-cultured with ACE2 expressing HEK293T cells for 16 hr before fluorescence imaging. ( B ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates collected from THP-1 supernatants-treated HEK293T-S and HEK293T-ACE2 described in (A) for 16 hr. Fetal bovine serum (FBS)-free RPMI 1640 served as medium control. Data are representative of six individual repeats and displayed as individual points with mean ± standard error of the mean (SEM). ( C ) Immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from THP-1 supernatants-treated HEK293T-S and HEK293T-ACE2 described in (A) for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( D ) Representative fluorescent image captured at 488 nm from THP-1 supernatants-treated HEK293T-S-ZsGreen and HEK293T-ACE2 for 16 hr. ( E ) Schematic presentation of THP-1 supernatants pre-treatment on authentic SARS-CoV-2-infected cells. Pre-treatment of HEK293T-ACE2 cells with THP-1 supernatants for 1 hr, then inoculated with 0.5 multiplicity of infection (MOI) Delta or wild-type (WT) authentic SARS-CoV-2 virus. Imaging was performed at 24 hr post-infection (hpi) before cell lysates were harvested for immunoblotting. ( F ) Immunoblots of Delta SARS-CoV-2 S, S2, cleaved S2’, N, and ACE2 proteins collected from HEK293T-ACE2 cells 24 hpi as described in (E). Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus Tubulin. ( G ) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’, and N proteins collected from Caco-2 cells 24 hpi as described in (E). Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus β-Actin. ( H ) Immunofluorescent images showing morphology of SARS-CoV-2-infected Caco-2 cells pre-treated with THP-1 supernatants. Anti-SARS-CoV-2 N was stained with Alexa Fluor 555, and nuclei were counterstained with DAPI, respectively. White arrowheads (D and H) indicate syncytia formation or infected cells, scale bars are indicative of 50 μm, and images are representative of three independent experiments. ( I ) Quantification of the infected area in (H). Figure 1—source data 1. Annotated, uncropped blots in . Figure 1—source data 2. Raw, uncropped blots in . Figure 1—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Cell Culture, Expressing, ATP Bioluminescent Assay, Western Blot, Fluorescence, Imaging, Luciferase, Activity Assay, Control, Infection, Virus, Staining

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Quantification of the fused area in . ( B ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates collected from TLR ligands-treated HEK293T-S and HEK293T-ACE2 for 16 hr. Data are representative of four individual repeats and displayed as individual points with mean ± standard error of the mean (SEM). ( C ) Immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from TLR ligands-treated HEK293T-S and HEK293T-ACE2 for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( D ) Representative fluorescent image captured at 488 nm from TLR ligands-treated HEK293T-S-ZsGreen and HEK293T-ACE2 for 16 hr. ( E ) Quantification of the infected area in (D). ( F ) Luciferase activity (RLU) measured from HEK293T cell lysates collected from PBMCs supernatants-treated HEK293T-S and HEK293T-ACE2 for 16 hr. 1% fetal bovine serum (FBS) RPMI 1640 served as medium control. Data are representative of five individual repeats and displayed as individual points with mean ± SEM. ( G ) Immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from PBMCs supernatants-treated HEK293T-S and HEK293T-ACE2 for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( H ) Representative fluorescent image captured at 488 nm from PBMCs supernatants-treated HEK293T-S-ZsGreen and HEK293T-ACE2 for 16 hr. White arrowheads (D and H) indicate syncytia formation. Scale bars, 50 μm. Images are representative of three individual repeats. ( I ) Quantification of the fused area in (H). Figure 1—figure supplement 1—source data 1. Annotated, uncropped blots in . Figure 1—figure supplement 1—source data 2. Raw, uncropped blots in . Figure 1—figure supplement 1—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Luciferase, Activity Assay, Western Blot, Infection, Control

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Immunoblots of wild-type (WT) SARS-CoV-2 S, S2, cleaved S2’, and N proteins collected from HEK293T-ACE2 cells 24 hr post-infection (hpi) as described in . Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus Tubulin. ( B, C ) Bright-field images of 0.5 multiplicity of infection (MOI) WT (B) or Delta (C) SARS-CoV-2-infected HEK293T-ACE2 cells pre-treated with THP-1 supernatants. White arrowheads indicate syncytia formation, scale bars are indicative of 50 μm, and images are representative of three independent experiments. Figure 1—figure supplement 2—source data 1. Annotated, uncropped blots in . Figure 1—figure supplement 2—source data 2. Raw, uncropped blots in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Western Blot, Infection

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Schematics of the cell-cell fusion model used to quantify spike-mediated syncytium formation upon treatment with different cytokines. Cells co-expressing SARS-CoV-2 spike and Cre were co-cultured with ACE2 and Stop-luc co-expressing HEK293T cells for 16 hr, before cell lysates were collected for bioluminescence assay and immunoblotting. Cells co-expressing SARS-CoV-2 spike and ZsGreen were co-cultured with ACE2 expressing HEK293T cells for 16 hr before fluorescence imaging. ( B ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates collected from different cytokines-treated HEK293T-S and HEK293T-ACE2 described in (A) for 16 hr. IL-1α (10 ng/mL), IL-1β (1 ng/mL), IL-6 (100 ng/mL), or IL-8 (100 ng/mL) were added into the cell-cell fusion system. Data are representative of six individual repeats and displayed as individual points with mean ± standard error of mean (SEM). ( C ) Luciferase activity (RLU) measured from HEK293T cell lysates collected from different concentrations of IL-1β-treated HEK293T-S and HEK293T-ACE2 for 16 hr. Data are representative of six individual repeats and displayed as individual points with mean ± SEM. ( D ) Immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from different concentrations of IL-1β-treated HEK293T-S and HEK293T-ACE2 for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( E ) Schematic presentation of IL-1β pre-treatment on authentic SARS-CoV-2-infected cells. Pre-treatment of HEK293T-ACE2 cells with different concentrations of IL-1β for 1 hr, then inoculated with 0.5 multiplicity of infection (MOI) Delta or wild-type (WT) authentic SARS-CoV-2 virus. Bright-field images were captured at 24 hr post-infection (hpi) before cell lysates were harvested for immunoblotting. ( F ) Immunoblots of Delta SARS-CoV-2 S, S2, cleaved S2’, N, and ACE2 proteins collected from HEK293T-ACE2 cells 24 hpi as described in (E). Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus Tubulin. ( G ) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’, and N proteins collected from Caco-2 cells 24 hpi as described in (E). Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus β-Actin. ( H ) Immunofluorescent images showing morphology of SARS-CoV-2-infected Caco-2 cells pre-treated with or without IL-1β. Anti-SARS-CoV-2 N was stained with Alexa Fluor 555, and nuclei were counterstained with DAPI, respectively. White arrowheads indicate syncytia formation or infected cells, scale bars are indicative of 50 μm and images are representative of three independent experiments. ( I ) Quantification of the infected area in (H). ( J ) Luciferase activity (RLU) measured from THP-1 supernatants-treated HEK293T-S and HEK293T-ACE2 in the presence or absence of IL-1 receptor antagonist (IL-1RA). Data are representative of four individual repeats and displayed as individual points with mean ± SEM. Figure 2—source data 1. Annotated, uncropped blots in . Figure 2—source data 2. Raw, uncropped blots in . Figure 2—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Expressing, Cell Culture, ATP Bioluminescent Assay, Western Blot, Fluorescence, Imaging, Luciferase, Activity Assay, Infection, Virus, Staining

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from different concentrations of IL-1α-treated HEK293T-S and HEK293T-ACE2 for 16 hr. Data and blots are representative of four individual repeats. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( B, C ) Representative fluorescent image captured at 488 nm from different concentrations of IL-1β (B) or IL-1α (C) treated HEK293T-S-ZsGreen and HEK293T-ACE2 for 16 hr. ( D ) Quantification of the fused area in (B). ( E ) Quantification of the fused area in (C). ( F ) Luciferase activity (RLU) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from IL-1α and IL-1β co-treated HEK293T-S and HEK293T-ACE2 for 16 hr. Data and blots are representative of three individual repeats. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( G ) Representative fluorescent image captured at 488 nm from IL-1α and IL-1β co-treated HEK293T-S-ZsGreen and HEK293T-ACE2 for 16 hr. White arrowheads (B, C, and G) indicate syncytia formation. Scale bars, 50 μm. Images are representative of three individual repeats. ( H ) Quantification of the fused area in (G). Figure 2—figure supplement 2—source data 1. Annotated, uncropped blots in . Figure 2—figure supplement 2—source data 2. Raw, uncropped blots in . Figure 2—figure supplement 2—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Luciferase, Activity Assay, Western Blot

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Immunoblots of wild-type (WT) SARS-CoV-2 S, S2, cleaved S2’, and N proteins collected from HEK293T-ACE2 cells 24 hr post-infection (hpi) as described in . Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus Tubulin. ( B, C ) Bright-field images of 0.5 multiplicity of infection (MOI) WT (B) or Delta (C) SARS-CoV-2-infected HEK293T-ACE2 cells pre-treated with different concentrations of IL-1β. White arrowheads indicate syncytia formation, scale bars are indicative of 50 μm, and images are representative of three independent experiments. Figure 2—figure supplement 3—source data 1. Annotated, uncropped blots in . Figure 2—figure supplement 3—source data 2. Raw, uncropped blots in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Western Blot, Infection

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A, B ) ELISA of IL-1β concentrations in supernatants from THP-1 (A) and peripheral blood mononuclear cells (PBMCs) (B) after Toll-like receptor (TLR) ligands stimulation. Data are representative of three individual repeats. ( C ) Immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from THP-1 supernatants-treated HEK293T-S and HEK293T-ACE2 in the presence or absence of IL-1 receptor antagonist (IL-1RA). Blots are representative of three individual repeats. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( D ) ELISA of IL-1β concentrations in supernatants from sgControl and sg TLR2 THP-1 cells after TLR ligands stimulation. Data are representative of four individual repeats. ( E ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates collected from sgControl and sg TLR2 THP-1 supernatants-treated HEK293T-S and HEK293T-ACE2 for 16 hr. Fetal bovine serum (FBS)-free RPMI 1640 served as medium control. Data are representative of three individual repeats and displayed as individual points with mean ± standard error of mean (SEM). ( F ) ELISA of IL-1β concentrations in supernatants from TAK1/IKKβ inhibitors-treated THP-1 cells after PGN stimulation. Data are representative of three individual repeats. ( G ) Luciferase activity (RLU) measured from HEK293T cell lysates collected from TAK1/IKKβ inhibitors pre-treated THP-1 supernatants added onto HEK293T-S and HEK293T-ACE2 cells for 16 hr. FBS-free RPMI 1640 served as medium control. Data are representative of three individual repeats and displayed as individual points with mean ± SEM. ( H ) ELISA of IL-1β concentrations in supernatants from TAK1/IKKβ inhibitors-treated PBMCs after PGN stimulation. Data are representative of three individual repeats. ( I ) Luciferase activity (RLU) measured from HEK293T cell lysates collected from TAK1/IKKβ inhibitors pre-treated PBMCs supernatants added onto HEK293T-S and HEK293T-ACE2 cells for 16 hr. 1% FBS RPMI 1640 served as medium control. Data are representative of three individual repeats and displayed as individual points with mean ± SEM. Figure 2—figure supplement 4—source data 1. Annotated, uncropped blots in . Figure 2—figure supplement 4—source data 2. Raw, uncropped blots in . Figure 2—figure supplement 4—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Activity Assay, Control

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Schematics of the cell-cell fusion model used to determine cell types affected by IL-1β. Pre-treated HEK293T-S or HEK293T-ACE2 cells or both with 1 ng/mL IL-1β for 6 hr, then co-cultured for 16 hr after washing with phosphate buffered saline (PBS). Cells co-expressing SARS-CoV-2 spike and Cre were co-cultured with ACE2 and Stop-luc co-expressing HEK293T cells for 16 hr, before cell lysates were collected for bioluminescence assay and immunoblotting. Cells co-expressing SARS-CoV-2 spike and ZsGreen were co-cultured with ACE2 expressing HEK293T cells for 16 hr before fluorescence imaging. ( B ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from different treatments of IL-1β described in (A) for 16 hr. Data and blots are representative of six individual repeats. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( C ) Immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from 1 ng/mL IL-1β-treated HEK293T-S and Vero E6-ACE2 for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( D ) Immunoblots showing full-length spike, S2, and cleaved S2’ collected from 1 ng/mL IL-1β-treated HEK293T-S and Calu-3 for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. Figure 2—figure supplement 5—source data 1. Annotated, uncropped blots in . Figure 2—figure supplement 5—source data 2. Raw, uncropped blots in . Figure 2—figure supplement 5—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Cell Culture, Saline, Expressing, ATP Bioluminescent Assay, Western Blot, Fluorescence, Imaging, Luciferase, Activity Assay

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Representative fluorescent image captured at 488 nm from HEK293T-S-ZsGreen co-cultured with HEK293T-ACE2, Vero E6-ACE2, and Calu-3 for 16 hr with or without 1 ng/mL IL-1β. White arrowheads indicate syncytia formation. Scale bars, 50 μm. Images are representative of three individual repeats. ( B ) Quantification of the fused area in (A). ( C ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates from 1 ng/mL IL-1β-treated HEK293T-S (S-Alpha, S-Beta, S-Delta, S-Omicron BA.4) and HEK293T-ACE2 for 16 hr. Data are representative of four individual repeats and displayed as individual points with mean ± standard error of mean (SEM). ( D, E ) Immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from 1 ng/mL IL-1β-treated HEK293T-S (S-Alpha, S-Beta, S-Delta (D), S-Omicron BA.4 (E)) and HEK293T-ACE2 for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. Figure 2—figure supplement 6—source data 1. Annotated, uncropped blots in . Figure 2—figure supplement 6—source data 2. Raw, uncropped blots in . Figure 2—figure supplement 6—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Cell Culture, Luciferase, Activity Assay, Western Blot

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Schematics of the cell-cell fusion model used to quantify SARS-CoV and MERS-CoV spike-mediated syncytia formation upon IL-1β treatment. Cells co-expressing SARS-CoV or MERS-CoV spike and Cre were co-cultured with ACE2 or DPP4 and Stop-luc co-expressing HEK293T cells for 16 hr, before cell lysates were collected for bioluminescence assay and immunoblotting. Cells co-expressing SARS-CoV or MERS-CoV spike and Cre were co-cultured with ACE2 or DPP4 and Stop-mCherry co-expressing HEK293T cells for 16 hr before fluorescence imaging. ( B ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates from 1 ng/mL IL-1β-treated HEK293T-S (SARS-S) and HEK293T-ACE2 for 16 hr. Data are representative of four individual repeats and displayed as individual points with mean ± SEM. ( C ) Luciferase activity (RLU) measured from HEK293T cell lysates from 1 ng/mL IL-1β-treated HEK293T-S (MERS-S) and HEK293T-DPP4 for 16 hr. Data are representative of four individual repeats and displayed as individual points with mean ± SEM. ( D ) Immunoblots showing SARS-CoV full-length spike, cleaved S2’ and ACE2 collected from 1 ng/mL IL-1β-treated HEK293T-S (SARS-S) and HEK293T-ACE2 for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( E ) Immunoblots showing MERS-CoV full-length spike, cleaved S2’ collected from 1 ng/mL IL-1β-treated HEK293T-S (MERS-S) and HEK293T-DPP4 for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( F ) Representative fluorescent images captured at 594 nm from 1 ng/mL IL-1β-treated HEK293T-S (SARS-S) and HEK293T-ACE2 or HEK293T-S (MERS-S) and HEK293T-DPP4 for 16 hr. White arrowheads indicate syncytia formation. Scale bars, 50 μm. Images are representative of three individual experiments. ( G ) Quantification of the fused area in (F). Figure 2—figure supplement 7—source data 1. Annotated, uncropped blots in . Figure 2—figure supplement 7—source data 2. Raw, uncropped blots in . Figure 2—figure supplement 7—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Expressing, Cell Culture, ATP Bioluminescent Assay, Western Blot, Fluorescence, Imaging, Luciferase, Activity Assay

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Schematics of gene knockout or inhibitor treatment in the IL-1 receptor pathway. ( B ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from HEK293T-S and HEK293T-ACE2 pre-treated with 1000 ng/mL IL-1 receptor antagonist (IL-1RA) for 30 min, then treated with 1 ng/mL IL-1β for 16 hr. Data and blots are representative of five individual repeats. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( C ) Luciferase activity (RLU) measured from cell lysates collected from 10 ng/mL IL-1α or 1 ng/mL IL-1β-treated sgControl or sg MyD88 HEK293T cell-cell fusion system for 16 hr. Data are representative of four individual repeats and displayed as individual points with mean ± SEM. ( D ) Luciferase activity (RLU) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2, cleaved S2’, and ACE2 collected from HEK293T-S and HEK293T-ACE2 pre-treated with 2 μM IRAK1/4 inhibitor for 30 min, then treated with 1 ng/mL IL-1β for 16 hr. Data and blots are representative of four individual repeats. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( E ) Luciferase activity (RLU) measured from cell lysates collected from 10 ng/mL IL-1α- or 1 ng/mL IL-1β-treated sgControl or sg TRAF6 HEK293T cell-cell fusion system for 16 hr. Data are representative of four individual repeats and displayed as individual points with mean ± SEM. ( F ) Immunoblots showing full-length spike, S2, cleaved S2’, and N collected from Caco-2 cells, which were pre-treated with 2 μM IRAK1/4 inhibitor and 10 ng/mL IL-1β for 1 hr, then infected with authentic SARS-CoV-2 for 24 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ or N versus β-Actin. ( G ) Immunoblots showing full-length spike, S2, cleaved S2’, and N collected from Calu-3 cells, which were infected with authentic SARS-CoV-2 for 1 hr, then washed with phosphate buffered saline (PBS) before treated with 2 μM IRAK1/4 inhibitor and 10 ng/mL IL-1β for 24 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ or N versus β-Actin. Figure 3—source data 1. Annotated, uncropped blots in . Figure 3—source data 2. Raw, uncropped blots in . Figure 3—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Gene Knockout, Luciferase, Activity Assay, Western Blot, Infection, Saline

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Immunoblots showing full-length spike, S2 and cleaved S2’, ACE2, and MyD88 collected from 10 ng/mL IL-1α- or 1 ng/mL IL-1β-treated sgControl or sg MyD88 HEK293T cell-cell fusion system for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( B ) Immunoblots showing full-length spike, S2 and cleaved S2’, ACE2, and TRAF6 collected from 10 ng/mL IL-1α- or 1 ng/mL IL-1β-treated sgControl or sg TRAF6 HEK293T cell-cell fusion system for 16 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. Figure 3—figure supplement 1—source data 1. Annotated, uncropped blots in . Figure 3—figure supplement 1—source data 2. Raw, uncropped blots in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Western Blot

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Luciferase activity (relative luminescence units [RLU]) measured from cell lysates and immunoblots showing full-length spike, S2 and cleaved S2’, ACE2 collected from 1 ng/mL IL-1β-treated sgControl or sg MAP3K7 HEK293T cell-cell fusion system for 16 hr. Data and blots are representative of five individual repeats. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( B ) Luciferase activity (RLU) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2 and cleaved S2’, ACE2 collected from HEK293T-S and HEK293T-ACE2 pre-treated with different concentrations of TPCA1 for 30 min, then treated with 1 ng/mL IL-1β for 16 hr. Data and blots are representative of three individual repeats. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( C ) mRNA levels of NF-κB pathway-related genes in HEK293T cells after 1 ng/mL IL-1β for 4 hr. Data are representative of three individual repeats. ( D ) Luciferase activity (RLU) measured from cell lysates collected from 1 ng/mL IL-1β-treated sgControl, sg RELB , sg NFKB1, or sg NFKBIA HEK293T cell-cell fusion system for 16 hr. Data are representative of four individual repeats and displayed as individual points with mean ± standard error of mean (SEM). Figure 3—figure supplement 2—source data 1. Annotated, uncropped blots in . Figure 3—figure supplement 2—source data 2. Raw, uncropped blots in . Figure 3—figure supplement 2—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Luciferase, Activity Assay, Western Blot

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Single-channel confocal images of . ( B ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2, cleaved S2’, ACE2, and Myc-RhoA collected from transfected vector, 10 or 20 ng constitutively active RhoA mutant (RhoA-CA) both in HEK293T-S and in HEK293T-ACE2 cells. Data and blots are representative of four individual repeats. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( C ) Single-channel confocal images of (top panel). ( D ) Single-channel confocal images of (middle panel). Figure 4—figure supplement 2—source data 1. Annotated, uncropped blots in . Figure 4—figure supplement 2—source data 2. Raw, uncropped blots in . Figure 4—figure supplement 2—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Luciferase, Activity Assay, Western Blot, Transfection, Plasmid Preparation, Mutagenesis

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Immunoblots of wild-type (WT) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S, S2, cleaved S2’, N, and Myc-RhoA collected from Caco-2 cells, which were transfected with vector, 10 or 20 ng RhoA-CA before infection with 0.5 multiplicity of infection (MOI) WT authentic SARS-CoV-2 for 24 hr. Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus β-Actin. ( B ) Representative confocal images of F-actin stained with phalloidin-488 from Caco-2 cells described in (A). Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells. White arrowheads indicate the enrichment or disappearance of F-actin, scale bars, 10 μm. Images are representative of four independent experiments. ( C ) Single-channel confocal images of (B). ( D ) Single-channel confocal images of . ( E ) Co-immunoprecipitation (IP) and input controls of full-length spike protein after anti-V5 or anti-IgG pull-down from cell lysates mixed between HEK293T cells expressing ACE2-V5-6his or Spike protein with vector or RhoA-CA (10 ng). Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S or S2 versus ACE2 in IP group and S, S2, or ACE2 versus Tubulin in input group. ( F ) Representative confocal images of wheat germ agglutinin (WGA, cell surface marker) and ACE2 from HEK293T cells co-transfected with ACE2 and vector or RhoA-CA (10 ng). Scale bars, 10 μm. Images are representative of three independent experiments. ( G ) Quantification of the relative ACE2 expression on the cell surface in (F). Figure 5—figure supplement 2—source data 1. Annotated, uncropped blots in . Figure 5—figure supplement 2—source data 2. Raw, uncropped blots in . Figure 5—figure supplement 2—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Western Blot, Transfection, Plasmid Preparation, Infection, Staining, Expressing, Immunoprecipitation, Marker

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Representative confocal images of wheat germ agglutinin (WGA) and ACE2 from HEK293T cells transfected with ACE2 and treated with phosphate buffered saline (PBS) or IL-1β. Scale bars, 10 μm. Images are representative of three independent experiments. ( B ) Quantification of the relative ACE2 expression on the cell surface in (A). ( C ) Representative confocal images of WGA and Spike from HEK293T cells transfected with Spike and treated with PBS or IL-1β. Scale bars, 10 μm. Images are representative of three independent experiments. ( D ) Quantification of the relative Spike expression on the cell surface in (C). ( E ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2 and cleaved S2’, ACE2 collected from HEK293T-S, and HEK293T-ACE2 pre-treated with different concentrations of Y-27632 for 30 min, then treated with 1 ng/mL IL-1β for 16 hr. Data and blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( F ) Immunoblots of wild-type (WT) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S, S2, cleaved S2’, and N collected from Caco-2 cells, which were treated with different concentrations of Y-27632 and 10 ng/mL IL-1β for 1 hr, then infected with 0.5 multiplicity of infection (MOI) WT authentic SARS-CoV-2 for 24 hr. Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus β-Actin. ( G ) Representative confocal images of F-actin stained with phalloidin-488 in Caco-2 cells described in (F). Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells. White arrowheads indicate the enrichment or disappearance of F-actin, scale bars, 10 μm. Images are representative of four independent experiments. Figure 5—figure supplement 3—source data 1. Annotated, uncropped blots in . Figure 5—figure supplement 3—source data 2. Raw, uncropped blots in . Figure 5—figure supplement 3—source data 3. Source data of the individual points in .

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Transfection, Saline, Expressing, Luciferase, Activity Assay, Western Blot, Infection, Staining

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal antibody (mAb) to HRP-conjugated β-tubulin , Abclonal , Cat#:AC030, RRID: AB_2769870 , WB (1:5000).

Techniques: Purification, In Vitro, In Vivo, Recombinant, Enzyme-linked Immunosorbent Assay, Activation Assay, Plasmid Preparation, Sequencing