Ab109131

Two lung cancer tissue microarrays were purchased from Superbiotek (Shanghai, PRC), containing 63 and 78 paired tumors and adjacent normal tissues from LUAD (LUC1601) and LUSC (LUC1602) patients, respectively. A summary of the clinical pathological information including subtype, tumor/nodal stage, histological grade, and information about patient follow-up. The tissue sections were stained with an anti-TMPRSS2 antibody (ab109131, ABCAM, United States) (dilution: 1: 1000). Following the general standard IHC staining methods, TMAs sections were used to measure the TMPRSS2 protein levels. The staining results were quantified according to the following criteria: for staining degree: 1, weak; 2, intermediate; 3, strong; For the proportion of positive cells: 1, 0–25%; 2, 26–50%; 3, 51–75%; 4, >75%. The staining score was obtained by multiplying staining degree by the proportion, 0–5 was considered low expression, and >5 was high expression.

Immunohistochemistry was performed on biopsies obtained from 6 NAFLD patients for which formalin-fixed liver sections were available, using the rabbit anti-human ACE2 (ab108252) and rabbit anti-human TMPRSS2 (ab109131, Abcam, Cambridge, UK), both diluted at 1:6400 according to the manufacturer’s instructions, and developed using DAB (3,3′-Diaminobenzidine, Vector Labs) as chromogen as previously described [22 (link)]. Histological images were taken using the BX41 microscope (Olympus), coupled with Leica LAS X Life Science software.

Human embryonic kidney 293T cells (HEK293T), African green monkey kidney cells Vero E6 and Vero 76 cells, murine macrophage RAW264.7, and murine fibroblast NIH3T3 cells were maintained at 37 °C in Dulbecco modified Eagle medium (DMEM; Gibco) supplemented with 10% fetal calf serum (FCS; Gibco), 1% GlutaMAX (Gibco), and 1% sodium pyruvate (100 mM; Gibco). A. albopictus C6/36 cells were grown at 28 °C in Royal Park Memorial Institute (RPMI) medium (Gibco, USA), 10% FCS (Gibco), and 1% GlutaMAX (200 mM; Gibco). Following viral infections, cells were maintained in corresponding media with 2% FCS and 10,000 U/mL of penicillin and 10,000 μg/mL of streptomycin (Gibco, USA).
Vero E6-TMPRSS2 cells were generated by transduction with lentivirus containing puromycin-selectable codon-optimized human TMPRSS2 construct and validated with anti-TMPRSS2 antibody (Abcam, ab109131) (full details are available in Supplementary Note 1). HEK293T-hACE2 cells were provided by Jesse Bloom (Fred Hutchinson Cancer Research Centre, Washington, USA)¹⁸ .

Antibodies against ACE2 (ab15348), TMPRSS2 (ab109131), and GAPDH (ab8245) were obtained from Abcam (Cambridge, UK). Western blot samples were prepared as described previously [66 (link)]. Proteins on nitrocellulose membranes were visualized with the biotin/streptavidin-horseradish peroxidase system (GE Healthcare; Little Chalfont, UK) in combination with the “Renaissance Western Blot Chemoluminescence Reagent” (Perkin Elmer; Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).

All paraffin wax blocks were cut at 4 μm and the neighboring sections were used. Slides were dewaxed and re-hydrated with xylene and different concentration of ethanol. Slides were immersed in sodium citrate buffer (pH 6.0) and boiled for 10 min in a microwave oven. After treatment with 3% hydrogen peroxide for 10 min, sections were blocked with 1% milk and reacted with first antibodies against ACE2 (tcna2043, 1:800 dilution; Taiclone, Taiwan) and TMPRSS2 (ab109131, 1:500 dilution; Abcam, USA) for overnight at 4 °C. After washes, sections were subsequently incubated with biotinylated secondary antibodies (K4065; Dako, USA) for 1 h at room temperature. Slides were stained using 3,3′-diaminobenzidine chromogen (DAB) solution and counterstained with haematoxylin, followed by mounting. We photographed all of the lung tissues in the slides by microscopy (Nikon, USA). About 4–6 measurements of 40X magnification for each subject sample (n = 4 per group) were analyzed by image-analysis system (Image Pro-Plus, Media Cybernetics, USA). The labeled images were based on RGB 8-bit resolution per channel parameters. The segmented areas in the images were filtered to count stained area. Mean density and area thresholds were automatically defined based in the assessed images. After application, the area labeled and their counting per image was obtained [21 ].

Severe acute respiratory syndrome coronavirus 2 nucleoprotein was co-stained with ACE2 and TMPRSS2, respectively. Anti-SARS-CoV-2 nucleoprotein antibody (Cat No. 40143-T62; Sino Biological), anti-TMPRSS2 antibody (ab109131; Abcam), and anti-ACE2 antibody (ab108252; Abcam) were used in this assay.