Journal: iScience
Article Title: Evolution of the SARS-CoV-2 spike protein in utilizing host transmembrane serine proteases
doi: 10.1016/j.isci.2025.113318
Figure Lengend Snippet: SARS-CoV-2 entry into A549 cells overexpressing ACE2 and different TTSPs (A) Lentiviral vectors were used to introduce matriptase (M), prostasin (P), hepsin (H) and Kallikrein 13 (KLK13) into A549 ACE2 cells. (A) After antibiotic selection, cells were lysed and proteins were resolved by SDS-PAGE and analyzed by Western blotting. Each protease was detected in A549 ACE2 cell lysates (50 μg of protein per lane) using anti-FLAG antibody (B) KLK13 mRNA was evaluated using PCR in positively transduced cells. Β-actin (ACTB) mRNA was used as an internal control. (B) Control A549 cells with ACE2 (Ctrl), A549 cells overexpressing both ACE2 and TMPRSS2 (TMPRSS2), ACE2 and KLK13 (KLK13), ACE2 and matriptase (Matriptase), ACE2 and prostasin (Prostasin), and ACE2 and hepsin (Hepsin) were transduced with HIV pseudoviruses decorated with VSV-G protein (VSV-G) or each SARS-CoV-2 variant S glycoprotein. After 72 h at 37°C, pseudovirus entry was measured by measurement of the luminescence signal in the cell lysates (relative light units [RLUs]/mL of lysate sample). The assay was performed twice, each time in triplicate ( N = 3), and average values with standard deviation are presented. (C) Control A549 cells with ACE2 (Ctrl), A549 cells overexpressing both ACE2 and TMPRSS2 (TMPRSS2), ACE2 and KLK13 (KLK13), ACE2 and matriptase (Matriptase), ACE2 and prostasin (Prostasin), and ACE2 and hepsin (Hepsin) were infected with a given isolate (at 800 TCID 50 per mL) for 2 h. At 96 h post inoculation, replication of SARS-CoV-2 was evaluated by RT-qPCR analysis, and the data are presented as RNA copy numbers/ml (mean ± SD). The p values were calculated by two-way ANOVA. (∗ p < 0.05). n = 6 biological replicates.
Article Snippet: A549 (ATCC CCL-185), A549 cells overexpressing ACE2 (A549 ACE2 ), A549 cells overexpressing both ACE2 and TMPRSS2 (A549 ACE2/TMPRSS2 ), Vero cells (ATCC: CCL-81) and HEK293T cells (ATCC CRL-3216) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose; Thermo Fisher Scientific, Warsaw, Poland) supplemented with 5% heat-inactivated fetal bovine serum (5% DMEM; Thermo Fisher Scientific), penicillin (100 U/mL; Thermo Fisher Scientific), and streptomycin (100 μg/mL; Thermo Fisher Scientific).
Techniques: Introduce, Selection, SDS Page, Western Blot, Control, Transduction, Variant Assay, Standard Deviation, Infection, Quantitative RT-PCR
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![SARS-CoV-2 entry into A549 cells overexpressing ACE2 and different TTSPs (A) Lentiviral vectors were used to introduce matriptase (M), prostasin (P), hepsin (H) and Kallikrein 13 (KLK13) into A549 ACE2 cells. (A) After antibiotic selection, cells were lysed and proteins were resolved by SDS-PAGE and analyzed by Western blotting. Each protease was detected in A549 ACE2 cell lysates (50 μg of protein per lane) using anti-FLAG antibody (B) KLK13 mRNA was evaluated using PCR in positively transduced cells. Β-actin (ACTB) mRNA was used as an internal control. (B) Control A549 cells with ACE2 (Ctrl), A549 cells overexpressing both ACE2 and TMPRSS2 (TMPRSS2), ACE2 and KLK13 (KLK13), ACE2 and matriptase (Matriptase), ACE2 and prostasin (Prostasin), and ACE2 and hepsin (Hepsin) were transduced with HIV pseudoviruses decorated with VSV-G protein (VSV-G) or each SARS-CoV-2 variant S glycoprotein. After 72 h at 37°C, pseudovirus entry was measured by measurement of the luminescence signal in the cell lysates (relative light units [RLUs]/mL of lysate sample). The assay was performed twice, each time in triplicate ( N = 3), and average values with standard deviation are presented. (C) Control A549 cells with ACE2 (Ctrl), A549 cells overexpressing both ACE2 and TMPRSS2 (TMPRSS2), ACE2 and KLK13 (KLK13), ACE2 and matriptase (Matriptase), ACE2 and prostasin (Prostasin), and ACE2 and hepsin (Hepsin) were infected with a given isolate (at 800 TCID 50 per mL) for 2 h. At 96 h post inoculation, replication of SARS-CoV-2 was evaluated by RT-qPCR analysis, and the data are presented as RNA copy numbers/ml (mean ± SD). The p values were calculated by two-way ANOVA. (∗ p < 0.05). n = 6 biological replicates.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6188/pmc12496188/pmc12496188__gr3.jpg)