Journal: medRxiv

Article Title: KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney

doi: 10.1101/2020.09.16.20190694

Figure Lengend Snippet: (a) Representative immunostaining of KIM-1, SARS-CoV-2 nucleocapsid protein and pan-cytokeratin (top two rows), immunostaining of KIM-1 and SARS-CoV-2 nucleocapsid protein (3rd row), and immunostaining of ACE2, KIM-1, pan-cytokeratin and surfactant protein (bottom 2 panels) in kidney biopsy samples from COVID-19 patients. DAPI staining marks the nuclei in this and other panels. Scale bars, 20 μm. (b) Immunostaining of KIM-1 and ACE2 in a post-mortem lung sample from COVID-19 patients. Scale bar, 20 μm. (c) Patient information for three COVID-19-associated AKI kidney biopsy samples. The patients’ ages were at a range of 45-60 years old. (d) Immunostaining of KIM-1 and ACE2 in representative post-mortem kidney biopsy samples from four COVID-19 patients. Scale bars, 20 μm. (e) Immunostaining of KIM-1 and ACE2 post ischemia-reperfusion injury (IRI) in mouse kidneys. Scale bars, 20 μm. (f) Immunohistochemistry staining of SARS-CoV-2 nucleocapsid protein in a COVID-19-associated AKI patient. Scale bars, 50 μm (left panel), 20 μm (right panel). (g) Immunostaining of SARS-CoV-2 nucleocapsid protein in a COVID-19 post-mortem kidney section from the patient where immunohistochemisty is shown in . Scale bar, 20 μm.

Article Snippet: 10 μL of Recombinant Human TIM-1/KIM-1/HAVCR1 Fc Chimera Protein (0.5 mg/mL, 9319-TM; R&D systems Inc, Minneapolis, MN) was added into the mixture.

Techniques: Immunostaining, Staining, Immunohistochemistry

Journal: medRxiv

Article Title: KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney

doi: 10.1101/2020.09.16.20190694

Figure Lengend Snippet: (a) Negative-stain electron micrograph of SARS-CoV-2 virosomes displaying the spike ectodomain. The spike His-tag was bind to the nickel-nitrilotriacetic acid (Ni-NTA) lipids. (b) Top: A549 cell internalization of DiI-labeled virosomes as compared to control empty liposomes with equivalent Dil (0.5 nM for each). Scale bars, 20 μm. Bottom: Quantification of internalized DiI-positive cells by flow cytometry. *p=0.017. (c) Internalization of DiI-labeled virosomes by A549 cells in the presence of control mouse IgG, anti-KIM-1 (3F4 and AKG7) or TW-37, a specific inhibitor for KIM-1, or control DMSO, immunostained with KIM-1 antibody (green). Scale bars, 20 μm. (d) Quantification of internalized DiI-labeled virosomes by A549 cells as measured by flow cytometry. * p=0.0334, ** p=0.0017. (e) ACE2 immunostaining after uptake of DiI-labeled virosomes into A549 cells. Scale bars, 20 μm. (f) Internalization of DiI-labeled virosomes by mouse primary lung epithelial cells from wild-type mice or KIM-1 Δmucin mice after pretreatment with control IgG, anti-KIM-1 IgG (AF1817) or TW-37 or control DMSO. Scale bars, 20 μm. (g) Quantification of internalized DiI-labeled virosomes by mouse primary lung epithelial cells treated as described in and measured by ImageJ. Virosome-positive areas were normalized to the average of wild-type cells treated with DMSO and control IgG. Ten fields were analyzed in two independent experiments. * p<0.0001. (h) Internalization of DiI-labeled virosomes by human alveoloids with or without TW-37. Scale bar: 100 μm.

Article Snippet: 10 μL of Recombinant Human TIM-1/KIM-1/HAVCR1 Fc Chimera Protein (0.5 mg/mL, 9319-TM; R&D systems Inc, Minneapolis, MN) was added into the mixture.

Techniques: Staining, Labeling, Liposomes, Flow Cytometry, Immunostaining

Journal: medRxiv

Article Title: KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney

doi: 10.1101/2020.09.16.20190694

Figure Lengend Snippet: (a) Internalization of DiI-labeled virosomes or control Dil-labeled empty liposomes by LLC-PK1 cells stably expressing KIM-1 or control pcDNA. Scale bars, 20 μm. (b) Internalization assay of DiI-labeled virosomes on LLC-PK1 cells stably expressing human KIM-1 pretreated with TW-37 or control DMSO. Scale bars, 20 μm. (c) Z-stack analysis of KIM-1-expressing LLC-PK1 cells treated with DiI-labeled virosomes with or without TW-37 by confocal microscopy. Arrowheads indicate the internalized DiI-labeled virosomes. Scale bars: 10 μm. (d) Internalization of DiI-labeled virosomes by LLC-PK1 cells stably expressing KIM-1 or control pcDNA in a microfluidic channel with constant flow (0.2 dyn/cm 2 ). Arrows indicate direction of flow. (e) After staining with an orange-color fluorescent dye, KIM-1 expressing cells and control pcDNA cells were mixed, co-cultured together, and exposed to GFP-tagged SARS-CoV-2 pseudovirus (CoV2-01, SARS-CoV-2-S(GFP)) for 12 hours. Red and Green signals were imaged 1.5 days after infection. (f) Immunostaining of KIM-1 (AKG7) and ACE2 in LLC-PK1 cells stably expressing human KIM-1 or control pcDNA. Scale bars, 20 μm. (g) Western blotting of ACE2, KIM-1 (cytoplasmic domain, Ab #195) and ERK1/2 for confirmation of equal loading of lanes on using extracts of LLC-PK1 cells stably expressing human KIM-1 or control pcDNA. Asterisks indicate the bands for each protein. (h) Immunostaining of KIM-1 and ACE2 on LLC-PK1 cells stably expressing human KIM-1. Arrows indicate that KIM-1 expression is located in cell surface. Scale bars, 10 μm. (i) Left: Internalization assay of DiI-labeled virosomes on LLC-PK1 cells stably expressing wild-type or mutant mouse KIM-1 (wild-type, AAND mutant of WNFD binding motif in Ig domain (amino acid residues 112-115) or WFAA mutant) or control pcDNA. Scale bars: 20 μm. Right: Quantification of internalized DiI-labeled virosomes measured by ImageJ. * p<0.0001.

Article Snippet: 10 μL of Recombinant Human TIM-1/KIM-1/HAVCR1 Fc Chimera Protein (0.5 mg/mL, 9319-TM; R&D systems Inc, Minneapolis, MN) was added into the mixture.

Techniques: Labeling, Liposomes, Stable Transfection, Expressing, Confocal Microscopy, Staining, Cell Culture, Infection, Immunostaining, Western Blot, Mutagenesis, Binding Assay

Journal: medRxiv

Article Title: KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney

doi: 10.1101/2020.09.16.20190694

Figure Lengend Snippet: (a) Left: DiI-labeled virosome and control empty liposome internalization by human renal epithelial tubuloids. Right: Immunostaining of KIM-1 (green) in DiI-labeled virosome-treated tubuloids. Scale bars, 20 μm. (b) Phase contrast images of human renal epithelial tubuloids treated with SARS-CoV-2 virosomes. Scale bars, 20 μm. (c) Internalization assay of DiI-labeled virosomes by human renal epithelial tubuloids infected by adenovirus expressing GFP-KIM-1 (left two panels) and immunostaining of KIM-1 and ACE2 on human renal epithelial tubuloids infected by adenovirus expressing GFP-KIM-1 or control GFP-β-GAL (right two panels). Scale bars, 20 μm. (d) Immunostaining of KIM-1 and ACE2 in KIM-1 infected tubuloids. Scale bar, 20 μm. (e) Internalization assay of human renal epithelial tubuloids infected with adenovirus expressing GFP-KIM-1 and exposed to varying dilutions of DiI-labeled virosomes. Scale bars, 20 μm. (f) Quantification by flow cytometry of ACE2 expression of tubuloids infected with adenovirus expressing GFP-KIM-1 or control GFP-β-GAL. *p<0.0001. (g) Virosome uptake and KIM-1 immunostaining in human kidney organoids, untreated (Control) or treated with cisplatin (5 µM for 48 hr), and subsequently exposed to DiI-labeled virosomes. Scale bar, 20 μm.

Article Snippet: 10 μL of Recombinant Human TIM-1/KIM-1/HAVCR1 Fc Chimera Protein (0.5 mg/mL, 9319-TM; R&D systems Inc, Minneapolis, MN) was added into the mixture.

Techniques: Labeling, Immunostaining, Infection, Expressing, Flow Cytometry

Journal: medRxiv

Article Title: KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney

doi: 10.1101/2020.09.16.20190694

Figure Lengend Snippet: (a) Flow cytometry-based binding assay between KIM-1 and SARS-CoV-2 Spike protein with or without TW-37. (b) Quantitative analysis of data in . * p=0.0029, ** p=0.0004. (c) 293 cells expressing KIM-1 without ACE2, ACE2 without KIM-1, and control pcDNA were exposed to SARS-CoV-2 and replicated virus were quantified by in vitro microneutralization assay. * p=0.0113, ** p=0.0027, *** p=0.0001, **** p<0.0001.

Article Snippet: 10 μL of Recombinant Human TIM-1/KIM-1/HAVCR1 Fc Chimera Protein (0.5 mg/mL, 9319-TM; R&D systems Inc, Minneapolis, MN) was added into the mixture.

Techniques: Flow Cytometry, Binding Assay, Expressing, Virus, In Vitro, Microneutralization Assay