Journal: bioRxiv

Article Title: IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2

doi: 10.1101/2024.12.24.630260

Figure Lengend Snippet: Primer-specific RT-qPCR for relative mRNA expression of mouse ACE2 (mACE2) (A), human ACE2 (hACE2) (B), and mouse ACE1 (mACE1) (C) from lung tissue homogenates of wild-type control (C57BL/6J) mice, K-18 promoter-driven hACE2 overexpression ( K18-hACE2 ) mice, and humanized ACE2 ( hACE2 ) mice (**, P<0.0005; ***, P<0.0001; compared to C57BL/6J by ANOVA; n=8, 4 male and 4 female mice). (D) Representative photomicrographs of hACE2 (red) expression in lung airways marked for alpha-tubulin (green) with corresponding quantitative data (E). Hoechst, blue. Bar, 200 microns. Data, mean ± SD.

Article Snippet: Membranes were blocked in fluorescent blocking buffer (Rockland Immunochemicals, MB070) and probed at 4°C overnight with specific antibodies directed against human ACE2 (0.4 μg/mL; R&D Systems, MAB10823) or Mouse ACE2 (0.25 μg/mL; R&D Systems, AF3437) or a species nonspecific ACE2 antibody (0.4 μg/mL; R&D Systems, AF933).

Techniques: Quantitative RT-PCR, Expressing, Control, Over Expression

Journal: bioRxiv

Article Title: IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2

doi: 10.1101/2024.12.24.630260

Figure Lengend Snippet: (A) BMDMs from control or myeloid IL-1β -deleted mice (mIL-1β KO) hACE2 mice were primed with or without LPS (10 ng/mL) and exposed to cholesterol crystals (0 or 1000 μg/mL) for 24 hours to induce inflammasome activation, followed by RT-qPCR for relative IL-1β mRNA expression (***, P<0.0001 compared to all others using ANOVA; n=6 mice total, 3 males and 3 females). (B) BMDMs treated as in (A), followed by ELISA on cell culture supernatants for secreted, mature IL-1β protein (***, P<0.0001 compared to all others using ANOVA; n=6 mice total, 3 males and 3 females). (C) BMDMs treated as in (A), followed by quantitative RT-qPCR for relative hACE2 mRNA expression (***, P<0.0001 compared to all others by ANOVA; n=6 mice total, 3 males and 3 females). (D) Quantitative PCR of the ACE2 promoter after ChIP, using antibody specific to p65 (RELA) relative to isotype control, of lysate from BMDMs stimulated with LPS+IFN-γ for 24 hours (***, P=0.0001 by t -test; n=6 mice total, 3 males and 3 females). (E) BMDMs from control or mIL-1β KO mice were treated with LPS+IFN-γ for 24 hours in the presence or absence of the NF-κB inhibitor, JSH23, at indicated concentrations, followed by RT-qPCR for relative hACE2 mRNA expression in cell lysates (***, P≤0.0001 compared to control at 0 concentration by ANOVA; n=6 mice total, 3 males and 3 females). (F) Volcano plot demonstrating differential RNA-seq transcriptome data from BMDMs cultured from hACE2 mice and stimulated with LPS+IFN-γ for 18 hours, followed by infection with SARS-CoV-2 for 24 hours (n=3 male mice). Dashed line, P adj =0.05. (G) Heatmap illustrating normalized counts for the top 20 differentially expressed genes derived from (F) with each column representing an individual mouse and each row representing mRNA from a single gene (n=3 male mice). Data, mean ± SD.

Article Snippet: Membranes were blocked in fluorescent blocking buffer (Rockland Immunochemicals, MB070) and probed at 4°C overnight with specific antibodies directed against human ACE2 (0.4 μg/mL; R&D Systems, MAB10823) or Mouse ACE2 (0.25 μg/mL; R&D Systems, AF3437) or a species nonspecific ACE2 antibody (0.4 μg/mL; R&D Systems, AF933).

Techniques: Control, Activation Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Concentration Assay, RNA Sequencing Assay, Infection, Derivative Assay

Journal: bioRxiv

Article Title: IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2

doi: 10.1101/2024.12.24.630260

Figure Lengend Snippet: Primer-specific RT-qPCR for relative mRNA expression of mouse ACE2 (mACE2) (A), human ACE2 (hACE2) (B), and mouse ACE1 (mACE1) (C) from lung tissue homogenates of wild-type control (C57BL/6J) mice, K-18 promoter-driven hACE2 overexpression ( K18-hACE2 ) mice, and humanized ACE2 ( hACE2 ) mice (**, P<0.0005; ***, P<0.0001; compared to C57BL/6J by ANOVA; n=8, 4 male and 4 female mice). (D) Representative photomicrographs of hACE2 (red) expression in lung airways marked for alpha-tubulin (green) with corresponding quantitative data (E). Hoechst, blue. Bar, 200 microns. Data, mean ± SD.

Article Snippet: Membranes were blocked in fluorescent blocking buffer (Rockland Immunochemicals, MB070) and probed at 4°C overnight with specific antibodies directed against human ACE2 (0.4 μg/mL; R&D Systems, MAB10823) or Mouse ACE2 (0.25 μg/mL; R&D Systems, AF3437) or a species nonspecific ACE2 antibody (0.4 μg/mL; R&D Systems, AF933).

Techniques: Quantitative RT-PCR, Expressing, Control, Over Expression

Journal: bioRxiv

Article Title: IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2

doi: 10.1101/2024.12.24.630260

Figure Lengend Snippet: (A) BMDMs from control or myeloid IL-1β -deleted mice (mIL-1β KO) hACE2 mice were primed with or without LPS (10 ng/mL) and exposed to cholesterol crystals (0 or 1000 μg/mL) for 24 hours to induce inflammasome activation, followed by RT-qPCR for relative IL-1β mRNA expression (***, P<0.0001 compared to all others using ANOVA; n=6 mice total, 3 males and 3 females). (B) BMDMs treated as in (A), followed by ELISA on cell culture supernatants for secreted, mature IL-1β protein (***, P<0.0001 compared to all others using ANOVA; n=6 mice total, 3 males and 3 females). (C) BMDMs treated as in (A), followed by quantitative RT-qPCR for relative hACE2 mRNA expression (***, P<0.0001 compared to all others by ANOVA; n=6 mice total, 3 males and 3 females). (D) Quantitative PCR of the ACE2 promoter after ChIP, using antibody specific to p65 (RELA) relative to isotype control, of lysate from BMDMs stimulated with LPS+IFN-γ for 24 hours (***, P=0.0001 by t -test; n=6 mice total, 3 males and 3 females). (E) BMDMs from control or mIL-1β KO mice were treated with LPS+IFN-γ for 24 hours in the presence or absence of the NF-κB inhibitor, JSH23, at indicated concentrations, followed by RT-qPCR for relative hACE2 mRNA expression in cell lysates (***, P≤0.0001 compared to control at 0 concentration by ANOVA; n=6 mice total, 3 males and 3 females). (F) Volcano plot demonstrating differential RNA-seq transcriptome data from BMDMs cultured from hACE2 mice and stimulated with LPS+IFN-γ for 18 hours, followed by infection with SARS-CoV-2 for 24 hours (n=3 male mice). Dashed line, P adj =0.05. (G) Heatmap illustrating normalized counts for the top 20 differentially expressed genes derived from (F) with each column representing an individual mouse and each row representing mRNA from a single gene (n=3 male mice). Data, mean ± SD.

Article Snippet: Membranes were blocked in fluorescent blocking buffer (Rockland Immunochemicals, MB070) and probed at 4°C overnight with specific antibodies directed against human ACE2 (0.4 μg/mL; R&D Systems, MAB10823) or Mouse ACE2 (0.25 μg/mL; R&D Systems, AF3437) or a species nonspecific ACE2 antibody (0.4 μg/mL; R&D Systems, AF933).

Techniques: Control, Activation Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Concentration Assay, RNA Sequencing Assay, Infection, Derivative Assay