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 replication in the presence of entry inhibitors A549 cells expressing both ACE2 and TMPRSS2 (A) or A549 cells expressing only ACE2 (B) were infected with a given isolate (at 800 TCID 50 per mL) for 2 h in the presence of 100 μM camostat (A), 10 μM E64D, or control PBS (A and B). 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; ns = not significant). 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: Expressing, Infection, Control, Quantitative RT-PCR

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

Journal: ACS Omega

Article Title: Exploring SARS-CoV‑2 Spike RBD Pockets as Targets for Generic Drugs: A Combined Computational, Biophysical, and Biological Approach

doi: 10.1021/acsomega.5c05175

Figure Lengend Snippet: In vitro antiviral activity of selected compounds. (A) Antiviral activity against VSV pseudotyped with the S protein of Wuhan-Hu-1 and cellular toxicity for the indicated compounds in VeroE6 cells ( n = 3). (B) Virus production of SARS-CoV-2 infection by the indicated compounds at 20 μM or remdesivir (30 μM) in either A549-ACE2 or VeroE6-TMPRSS2 cells. (C) Antiviral activity of fingolimod against VSV pseudotyped with the indicated S protein ( n = 3). (D) Concentration reducing 50% of viral infection of VSV pseudotyped with the indicated S protein. All data represent the mean and standard error of 3 independent replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by two-tailed t test on log-transformed data.

Article Snippet: VeroE6-TMPRSS2 (Catalog No. JCRB1819, Japanese Collection of Research Bioresources), VeroE6 (kindly provided by Dr. Luis Enjuanes, CNB–CSIC, Spain), and A549-Ace2-TMPRSS2 (Catalog No. a549-hace2tpsa, Invivogen) were cultured in DMEM high glucose, with glutamine, 10% FBS, and 1% penicillin/streptomycin.

Techniques: In Vitro, Activity Assay, Virus, Infection, Concentration Assay, Two Tailed Test, Transformation Assay