Journal: EMBO Reports

Article Title: TMPRSS2-induced Golgi disruption restricts the incorporation of virus envelope glycoproteins into virions

doi: 10.1038/s44319-026-00797-2

Figure Lengend Snippet: ( A ) Infectivity of CoV-2-S pseudotyped lentiviruses was determined in ACE2- and TMPRSS2-WT (TM2-WT), TMPRSS2-WT-mCherry- (TM2-WT-Cherry)-, TMPRSS2-dCT-mCherry- (TM2-dCT-Cherry)-, TMPRSS2-S441A-mCherry- (TM2-S441A-Cherry)-, and/or TMPRSS2-WT-Wasabi- (TM2-WT-Wasabi)-expressing 293T cells (left and middle, the quantified results from a representative experiment in the two experiments independently conducted are expressed as mean ± SEM, n = 6). Infectivity of CoV-2-S pseudotyped lentiviruses was determined in ACE2- and TM2-WT-Cherry (with indicated ratio of plasmids) expressing 293 T cells (right, the quantified results are expressed as mean ± SEM of three separate experiments, n = 3). Representative data from three independent experiments are shown. ( B ) Immunoblotting analysis of cell lysate from 293 T cells expressing CoV-2-S (left) or HIV-1 Env (right) and TM2-WT-mCherry (TM2-WT) or TM2-S441A-mCherry (TM2-S441A). The ratio of CoV-2-S2 or gp160 to beta-actin was calculated based on band density (the quantified results are expressed as mean ± SEM of three separate experiments, n = 3). ( C ) Fluorescence microscopy analysis of A549-ACE2 and A549-ACE2-TMPRSS2-mCherry- (TM2)-expressing cells. ( D ) Infectivity of SARS-CoV-2 spike protein pseudotyped lentiviruses in ACE2- or ACE2- and TM2-expressing A549 cells (the quantified results from a representative experiment in the two experiments independently conducted are expressed as mean ± SEM, n = 4). ( E ) q-PCR analysis for TMPRSS2 (TM2) mRNA in indicated cell lines (A549-ACE2-TM2: A549-ACE2 cells stably expressing TM2; 293T-TM2: 293T cells transiently expressing TM2) (the quantified results from a representative experiment in the two experiments independently conducted are expressed as mean ± SEM, n = 3). ( F ) q-PCR analysis for TM2 mRNA in original Caco-2 (Caco-2 WT) and TM2 knockdown Caco-2 (Caco-2 TM2-KD) cells (the quantified results from a representative experiment in the two experiments independently conducted are expressed as mean ± SEM, n = 3). Scale bars indicate 50 μm. The results of Student’s t tests are shown above the bars.

Article Snippet: Human ACE2-expressing A549 , InvivoGen , a549-hace2.

Techniques: Infection, Expressing, Western Blot, Fluorescence, Microscopy, Stable Transfection, Knockdown

Journal: EMBO Reports

Article Title: TMPRSS2-induced Golgi disruption restricts the incorporation of virus envelope glycoproteins into virions

doi: 10.1038/s44319-026-00797-2

Figure Lengend Snippet: ( A ) Experimental scheme and luciferase activity for the infectivity of SARS-CoV-2-Spike (CoV-2-S) or vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviruses produced in CD4-mCherry- (Control)-, TMPRSS2-mCherry- (TM2-WT)-, TMPRSS2-dCT-mCherry- (TM2-dCT)-, or TMPRSS2-S441A-mCherry-(TM2-S441A)-expressing 293T cells. The infectivity of pseudotyped lentiviruses (3 ng/well) normalized with p24 amount (left in panels, the quantified results are expressed as mean ± SEM of three separate experiments, n = 3) was determined in ACE2- and TM2-expressing 293T cells (right in panels, the quantified results from a representative experiment in the three experiments independently conducted are expressed as mean ± SEM, n = 6 for CoV-2-S, n = 5 for VSV-G). ( B ) Experimental scheme (left) and plaque-formation assay using Vero E6 TMPRSS2 cells to test the infectivity of SARS-CoV-2 produced in A549-ACE2 or A549-ACE2 stably expressing TMPRSS2 (A549-ACE2-TM2) cells (middle, n = 3). A549-ACE2 or A549-ACE2-TM2 cells infected with SARS-CoV-2. After normalization with SARS-CoV-2 nucleocapsid protein (NP, left in panels, the quantified results are expressed as mean ± SEM of four separate experiments, n = 4) amounts, the viruses were used in a plaque-formation assay using Vero E6 TMPRSS2 cells. Plaque-forming unit (pfu) per 80 pg of NP is shown (right, the quantified results from a representative experiment in the three experiments independently conducted are expressed as mean ± SEM, n = 6). ( C ) Experimental scheme (top) and luciferase activity for the infectivity of CoV-2-S pseudotyped lentiviruses produced in Calu-3 cells. Calu-3 cells transfected with CoV-2-S and lentiviral plasmids were treated with the indicated concentrations of nafamostat. After normalization with p24 amounts (bottom left, the quantified results are expressed as mean ± SEM of five separate experiments, n = 5), the pseudotyped lentiviruses (1.0 pg/well) were used to infect 293T cells expressing ACE2 (293T-ACE2), but not TM2, and the infectivity was determined as luciferase activity (bottom right, the quantified results from a representative experiment in the two experiments independently conducted are expressed as mean ± SEM, n = 5). ( D ) Immunoblotting analysis of CoV-2-S pseudotyped lentiviruses in 293T cells expressing CD4-mCherry- (Control)-, TM2-WT, TM2-dCT, or TM2-S441A. The ratio of CoV-2-S2 to p24 was calculated based on band density (right, the quantified results are expressed as mean ± SEM of three separate experiments, n = 3). The samples were obtained from the same experiment, and the gels and blots were processed in parallel. ( E ) Experimental scheme (left top) and plaque-formation assay of SARS-CoV-2 produced in nafamostat-treated Caco-2 cells (left bottom, n = 3). Caco-2 cells infected with SARS-CoV-2 were treated with the indicated concentrations of nafamostat. After the 96 hr culture, viruses were purified and normalized with SARS-CoV-2 NP amounts (right top, the quantified results are expressed as mean ± SEM of six separate experiments, n = 6), the viruses were used in a plaque-formation assay using Vero E6 TMPRSS2 cells. Plaque-forming unit (pfu) per 25 pg of NP is shown (right bottom, the quantified results from a representative experiment in the three experiments independently conducted are expressed as mean ± SEM, n = 8). ( F ) Experimental scheme (top) and plaque-formation assay (middle) of SARS-CoV-2 produced in Caco-2 cells ( n = 3). Original (WT) and TM2-knockdown (KD) Caco-2 cells were infected with SARS-CoV-2. After the 96 hr culture and normalization with SARS-CoV-2 NP amounts (bottom left, the quantified results are expressed as mean ± SEM of three separate experiments, n = 3), the viruses were used in a plaque-formation assay using Vero E6 TMPRSS2 cells. Plaque-forming unit (pfu) per 100 pg of NP is shown (right bottom, the quantified results from two experiments among the three experiments independently conducted are expressed as mean ± SEM, n = 6). The number in vectors indicates the ratio of plasmid used for transfection. The results of Student’s t tests are shown above the bars. .

Article Snippet: Human ACE2-expressing A549 , InvivoGen , a549-hace2.

Techniques: Luciferase, Activity Assay, Infection, Virus, Produced, Control, Expressing, Plaque Formation Assay, Stable Transfection, Transfection, Western Blot, Purification, Knockdown, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: Collectin-11, a complement pattern recognition molecule, mediates pulmonary SARS-CoV-2 neutralization and protection

doi: 10.1371/journal.ppat.1014216

Figure Lengend Snippet: (A-C) Neutralization of infectious SARS-CoV-2 following incubation with two-fold dilutions of rCL-11 (black) in A549-hACE-2 cells (starting at 40 µg/ml) (A) , Calu-3 cells (starting at 20 µg/ml) (B) , and Huh7.5 cells (starting at 20 µg/ml) (C) using the conventional experimental setup. The mAb clone 61 was used as a neutralization control (orange) and rCL-11 buffer (gray) in corresponding dilutions was used to monitor buffer-mediated effects on the cells. The percentage of protected cells was determined from the number of SARS-CoV-2 infected cells in experimental wells compared to the number of infected cells in virus-only control wells after anti-S protein immunostaining. Data are shown as means ± SD of four (A549-hACE-2 and Calu-3 cells) or six (Huh7.5 cells) replicates.

Article Snippet: The human lung adenocarcinoma A549-hACE-2 cell line (InvivoGen, San Diego, California, USA) (A549 cells expressing human ACE-2) was maintained as described previously [ ].

Techniques: Neutralization, Incubation, Control, Infection, Virus, Immunostaining