Fig 1: Cell surface fucosylation mediates the binding/entry of TcsH.a The sensitivities of MCF-7 TMPRSS2‒/‒, GMDS‒/‒, FUT4‒/‒, and SLC35C1‒/‒ cell lines to TcsH were measured using the cytopathic cell-rounding assay. Error bars represent mean ± s.d., n = 6. b Schematic view of biosynthesis of fucosylated glycans, genes identified from the screens were highlighted. c Flow cytometry profiles of fluorescein isothiocyanate (FITC)-conjugated LTL (left) or AAL (right) binding to MCF-7 WT and KO cells. d The absence of TMPRSS2 expression in the MCF-7 TMPRSS2‒/‒ cells was validated by Western blot analysis. The MCF-7 GMDS‒/‒, FUT4‒/‒, and SLC35C1‒/‒ cells have similar TMPRSS2 expression levels compared to the WT cells. The experiments have been repeated independently twice with similar results. e Confocal fluorescence images show Rhodamine-labeled TcsH (green) or GFP-TcsH1832–2618 (green) bindings to the MCF-7 WT, GMDS‒/‒, FUT4‒/‒, SLC35C1‒/‒, and TMPRSS2‒/‒ cells, respectively. Cell nuclei were stained by Hoechst (blue). The scale bar represents 50 μm. f Co-incubation of the AAL (8 μg/mL) with TcsH (10 pM, 3.5 h) protected MCF-7 cells from intoxication and prevented cell rounding. g Confocal fluorescence images show binding of different GFP-fused TcsH CROPs fragments to the MCF-7 cells. Cell nuclei were stained by Hoechst (blue). The scale bar represents 50 μm.
Fig 2: ACE2 and TMPRSS2 transcript variants for the cell lines.A: Different exon usage of ACE2 for the top 10 ACE2 expressing cell lines plus CALU-3 (see S2 Fig for the top 25 cells). BFTC-905, CAL-33, CAL-27, M-07e, and MOLM-16 show reduced exon expression from exon 1–9 (blue rectangles). Cell lines are ordered alphabetically. ACE2 is reversely oriented. B: Missense variants in the coding regions of TMPRSS2 (V197M = rs12329760, G6V = rs75603675) for the selected cell lines. No mutations were found in the coding regions of ACE2. 0/1 denotes heterozygous and 1/1 homozygous mutations.
Fig 3: ACE2 and TMPRSS2 gene expression data for 301 DSMZ human cell lines in the CCLE RNA-seq data set.A: Expression of ACE2 and TMPRSS2 for selected disease entities. RNA-seq data were normalised and calculated to FPKM values. Gene expression for ACE2 and TMPRSS2 varies between and within the different tumour species. B: Expression of ACE2, TMPRSS2 and NRP1, cell lines sorted to ACE2 expression levels. Top 25 ACE2 expressing cell lines were selected for further studies, plus CALU-3 and two further neuronal cell lines with high NRP1 levels. FPKM—fragments per kilobase million: normalised gene expression data.
Fig 4: Autopsy results. A SARS-CoV-2 RNA copies in each organ system measured as log10 /10.000 cells. B–D Histopathology of the lung: areas of pneumonic congestion without any further destruction of the lung parenchyma (B PAS reaction, C, D H&E stain). E–G: SARS-CoV-2 in-situ-hybridization of the lung: specific reaction in pulmonary macrophages (E) and pneumocytes (F, G). H–J Histopathology of the liver: subtotal liver cirrhosis with moderate portal inflammatory infiltrates and proliferating bile ducts (H, arrow), areas of acute liver cell necrosis (I, asterisk) and cholestasis (J, arrow). K–M Immunohistochemistry of the liver: strong reactivity of bile duct epithelium for antibodies against ACE2 (K), Cathepsin L (L) and TMPRSS2 (M). N–P SARS-CoV-2 in-situ-hybridization of the liver: strong cytoplasmatic signals in the bile duct epithelium (arrows)
Fig 5: COVID-19 infection causes direct damage in the kidney. (A) Serial sections (5 μm thick) were prepared for H&E staining and IHC staining. H&E staining was used to distinguish the proximal tubules from the distal tubules. IHC staining was used to detect the related molecules of classical kidney injury in 10 COVID-19 patients’ kidney samples and 3 normal kidney samples. (B) The immunoreactive score (IRS) measured the expression of ACE2, TMPRSS2, TIM1, IL-6, and MCP-1 in normal and COVID-19 patients’ kidney samples. (C) The IRS measured the expression of ACE2, TMPRSS2, TIM1, IL-6, and MCP-1 in the distal tubule and proximal tubule of COVID-19 patients. Bar graphs show mean ± SD, Student’s t-test, p < 0.05 was statistically significant; NS, no significance.
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