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

Journal: Scientific Reports

Article Title: Altered plasma levels of the SARS-CoV-2-related proteins ACE2 and TMPRSS2 in patients with Crohn’s disease

doi: 10.1038/s41598-024-81810-3

Figure Lengend Snippet: Characterization of ACE2 and TMPRSS2 species in human plasma. ( A ) Schematic representation of ACE2 as a transmembrane type I protein and of the epitopes recognized by the antibodies used for it characterization (not drawn to scale). The carboxypeptidase and the transmembrane (TM) domains are represented. ( B ) Representative plasma samples from non-disease controls were immunoblotted with the antibodies AF933 (ectodomain) and the ab15348 (C-terminus). The C-terminus antibodies only recognize the full-length ACE2 which retains the C-terminal domain. The 70 and 75 kDa ACE2 immunoreacrive species lacking the C-terminal domain were identified as cleaved fragments, whereas 95, 100, 130 and 150 kDa were assigned as full-length forms. ( C ) Schematic representation of TMPRSS2 as a transmembrane type II protein and of the epitopes recognized by the antibodies used for it characterization (not drawn to scale). The peptidase and the transmembrane (TM) domains are represented, and the localization of the interdomain disulfide bond that served to link to the membrane-tethered prodomain fragment is indicated. ( D ) Plasma samples from control individuals (not the same than in B) were immunoblotted with the 14437-1-AP, an antibody that targets full-length TMPRSS2, recognizing a 55-kDa band and recognizes also the 35 kDa membrane-tethered prodomain fragments (black arrowhead) and the 25 kDa peptidase ectodomain (grey arrowhead). The same samples were also blotted with H00007113 (C-terminus) or the OAAB04388 (N-terminus) antibodies, confirming the identity of peptidase and prodomain fragments. (*) A smaller ~ 22-kDa band was attributed to a nonspecific reactivity, since it was immunoreactive to both the anti-C-terminal antibody and the anti-N-terminal antibody.

Article Snippet: Then, the membrane was blocked with Odyssey Blocking Buffer (PBS) and incubated for the 805 amino acids protein ACE2 with the antibodies raised against the ectodomain (amino acid residues 18–740) AF933 (R&D Systems; polyclonal goat, 1:200 dilution) or alternatively with the anti-C-terminus (immunogen: synthetic peptide corresponding to amino acid residues 788–805) antibody ab15348 (Abcam; rabbit polyclonal; 1:500 dilution).

Techniques: Membrane, Control

Journal: Scientific Reports

Article Title: Altered plasma levels of the SARS-CoV-2-related proteins ACE2 and TMPRSS2 in patients with Crohn’s disease

doi: 10.1038/s41598-024-81810-3

Figure Lengend Snippet: Levels of ACE2 and TMPRSS2 species in plasma from individuals affected by CD prior to treatment onset and from healthy controls. ( A ) Representative plasma samples from healthy controls (Ctrl; n = 10) and from patients with CD ( n = 10) were immunoblotted with the anti-ACE2 antibody AF933 (epitope: ectodomain). ( B ) The densitometric quantification of the cleaved 70 and 75 kDa ACE2 fragments, and full-length 95, 100 and 130 and 150 kDa species are shown. The relation between the 75 kDa fragment and the 100 kDa full-length was obtained, for each sample, by dividing the level of immunoreactivity of the 75-kDa band by the level of immunoreactivity of the 100-kDa band. The quotient (75 kDa/100 kDa) is represented. ( C ) The same plasma samples from controls and CD patients were immunoblotted with the anti-TMPRSS2 antibody 14437-1-AP (epitope: full-length protein). ( D ) Densitometric quantification of the full-length species (55 kDa), the peptidase fragment (25 kDa), and the prodomain fragment (35 kDa) are shown. The quotients between the prodomain fragment and the full-length species (35 kDa/55 kDa) are also shown. The figure shows the means ± SEM; the significant P value are also indicated.

Article Snippet: Then, the membrane was blocked with Odyssey Blocking Buffer (PBS) and incubated for the 805 amino acids protein ACE2 with the antibodies raised against the ectodomain (amino acid residues 18–740) AF933 (R&D Systems; polyclonal goat, 1:200 dilution) or alternatively with the anti-C-terminus (immunogen: synthetic peptide corresponding to amino acid residues 788–805) antibody ab15348 (Abcam; rabbit polyclonal; 1:500 dilution).

Techniques:

Journal: Scientific Reports

Article Title: Altered plasma levels of the SARS-CoV-2-related proteins ACE2 and TMPRSS2 in patients with Crohn’s disease

doi: 10.1038/s41598-024-81810-3

Figure Lengend Snippet: Levels of ACE2 and TMPRSS2 species in plasma from individuals affected by CD under biological or non-biological therapies. Plasma samples from healthy controls (Ctrl; n = 17) and from CD patients treated with azathioprine (Azath), infliximab (Inflix), adalimumab (Adal), or ustekinumab (Ustek), were immunoblotted with the anti-ACE2 antibody AF933 ( A ), or with the anti-TMPRSS2 antibody 14437-1-AP ( B ). Representative blots are shown. The densitometric quantification of the ACE2 and TMPRSS2 species were determined, and the ACE2 75 kDa/100 kDa and the TMPRSS2, 35 kDa/55 kDa, quotients were estimated as described in Fig. . The figure shows the means ± SEM; P values are also shown (ns: non-significant).

Article Snippet: Then, the membrane was blocked with Odyssey Blocking Buffer (PBS) and incubated for the 805 amino acids protein ACE2 with the antibodies raised against the ectodomain (amino acid residues 18–740) AF933 (R&D Systems; polyclonal goat, 1:200 dilution) or alternatively with the anti-C-terminus (immunogen: synthetic peptide corresponding to amino acid residues 788–805) antibody ab15348 (Abcam; rabbit polyclonal; 1:500 dilution).

Techniques: