Journal: Virulence
Article Title: The ApoA-I mimetic peptide 4F attenuates in vitro replication of SARS-CoV-2, associated apoptosis, oxidative stress and inflammation in epithelial cells
doi: 10.1080/21505594.2021.1964329
Figure Lengend Snippet: Impact of 4 F on cellular mediators involved in SARS-CoV-2 replication in lung epithelial cells. Calu3 cells were uninfected (mock) or infected with SARS-CoV-2 (MOI 0.1) and were treated with media alone (vehicle), or 4 F (10 μM) as in methods. Confluent cells were fixed 48 h post-infection (hpi) followed by processing for staining (flow cytometry) for mediators of viral entry (CD147), oxidative stress [mitochondrial reactive oxygen species (mito-ROS), Heme oxygenase 1 (HO-1)] and antiviral responses ([HO-1, myxovirus resistance MX Dynamin Like GTPase 1 (MX1)]. Flow cytometry was used in Calu3 cells to determine the median fluorescence intensity (MFI) of each target compared to a negative cell population (fluorescence minus one negative control for staining shown in light gray) (∆MFI). Representative data from three independent experiments are shown A. Flow cytometric staining for CD147 at 48 hours post-infection. B. Flow cytometric staining for the fluorochrome MitoSOX Red as a measure of mitochondrial reactive oxygen species (mito-ROS) content in Calu3 cells at 48 hours post-infection. C. Flow cytometric staining at 48 hours post-infection for HO-1. D. Flow cytometric staining at 48 hours post-infection for MX1
Article Snippet: The following antibodies were added to each tube and incubated in the dark for 20 minutes on ice: PE/Cyanine7 mouse anti-human CD147 antibody (clone HIM6 from Biolegend, San Diego, CA), mouse anti-human HO-1 antibody (clone HO-1-2 from Enzo Life Sciences) conjugated with Mix-n-Stain CF647 Antibody Labeling Kit from Biotium Inc (Hayward, CA), rabbit anti-human MX1 polyclonal antibody (from Proteintech, 13,750-1-AP), rabbit anti-SARS-CoV-2 (2019-nCoV) Spike S1 Antibody (Clone R007, Cat# 40,150-R007 from Sino Biologicals conjugated with Mix-n-Stain CF488 Antibody Labeling Kit from Biotium Inc (Hayward, CA), mouse anti-SARS-CoV/SARS-CoV-2 spike S antibody (clone 1A9 from GeneTex), Alexa Fluor® 647 Cleaved Caspase-3 (Asp175) (Clone D3E9 from Cell Signaling Technology, Cat# 9602S), Alexa Fluor® 647 Endorepellin/Perlecan/Heparan Sulfate Proteoglycan Antibody (clone A7L6 cat# NBP2-44,448).
Techniques: Infection, Staining, Flow Cytometry, Fluorescence, Negative Control
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![Impact of 4 F on cellular mediators involved in SARS-CoV-2 replication in lung epithelial cells. Calu3 cells were uninfected (mock) or infected with SARS-CoV-2 (MOI 0.1) and were treated with media alone (vehicle), or 4 F (10 μM) as in methods. Confluent cells were fixed 48 h post-infection (hpi) followed by processing for staining (flow cytometry) for mediators of viral entry (CD147), oxidative stress [mitochondrial reactive oxygen species (mito-ROS), Heme oxygenase 1 (HO-1)] and antiviral responses ([HO-1, myxovirus resistance MX Dynamin Like GTPase 1 <t>(MX1)].</t> Flow cytometry was used in Calu3 cells to determine the median fluorescence intensity (MFI) of each target compared to a negative cell population (fluorescence minus one negative control for staining shown in light gray) (∆MFI). Representative data from three independent experiments are shown A. Flow cytometric staining for CD147 at 48 hours post-infection. B. Flow cytometric staining for the fluorochrome MitoSOX Red as a measure of mitochondrial reactive oxygen species (mito-ROS) content in Calu3 cells at 48 hours post-infection. C. Flow cytometric staining at 48 hours post-infection for HO-1. D. Flow cytometric staining at 48 hours post-infection for MX1](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7485/pmc08437485/pmc08437485__KVIR_A_1964329_F0003_OC.jpg)