Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: a SARS-CoV-2 replication kinetics in HAE from different donors, HCoV-NL63 was used as a control ( n = 3). b Transepithelial electrical resistance (TEER in Ω cm 2 ) between the apical and basal poles was measured at each time point ( n = 3). c SARS-CoV-2 infected both ciliated cells (72 h pi) and secretory cells (72 h pi). arrows: virus particles, arrowhead: cilium, asterisk: secretory vesicle, insets dashed-line squares indicate magnification of arrowed areas. d Costaining of SARS-CoV-2 N protein (green) with ciliated cell marker β-tubulin-IV (red), goblet cell marker Muc5AC (red), club cell marker CCSP (red), and ACE2 (red) positive cells. HCoV-NL63 N protein (green) staining was used as a control (72 h pi). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Data a , b are the means ± s.d. of three independent biological replicates. Source data a – d are provided as a Source Data file.

Article Snippet: ACE2 , Bioss (bs-1004R), rabbit polyclonal (1:100).

Techniques: Infection, Marker, Staining

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: Source of antibodies and dyes with work concentration for immunofluorescence.

Article Snippet: ACE2 , Bioss (bs-1004R), rabbit polyclonal (1:100).

Techniques: Concentration Assay, Immunofluorescence

Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet: Stringent ACE2 requirement for pancreatic islet cell infection with SARS-CoV-2 (A) Representative contour plots gated on live α, β, and “other” cells pre-treated with IgG (irrelevant polyclonal goat antibody AF7197) or the anti-ACE2 blocking antibody AF933 prior to SARS-CoV-2 infection (48 h). (B) Summary of SARS-CoV-2 NP expression by live islet cell subsets as a function of IgG treatment or ACE2 blockade (n = 6 donors). (C) Percent infection inhibition for β and “other” cells (inhibition for α cells is not shown because the very low extent of α cell infection in IgG-treated cultures for 2 of 6 donors substantially skews such calculations). (D) Infectious SARS-CoV-2 titers and extent of infection inhibition following ACE2 blockade (n = 3 donors). (E) Quantification of chemokines and cytokines in UV-inactivated TCS of SARS-CoV-2-infected islet cell cultures under conditions of IgG treatment or ACE2 blockade (48-h infection, n = 3 donors). (F) Infectious SARS-CoV-2 titers in TCS as a function of glucose concentration in islet culture medium (n = 3 donors). (G) Quantification of CXCL10 and CXCL11 in TCS as a function of glucose concentration. All summary bar diagrams represent mean ± SD and scatter for the indicated number of donors; statistical analyses were conducted by paired t test or repeated-measures ANOVA with Tukey’s multiple comparisons where applicable (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). All summary bar diagrams represent mean ± SD and scatter for the indicated number of donors; statistical analyses were conducted by paired t test or repeated-measures ANOVA with Tukey’s multiple comparisons where applicable.

Article Snippet: Anti-human ACE2 (polyclonal goat IgG Poly5036) - AF647 (in-house conjugation) , Biolegend , Cat# 503602; RRID: AB_2892475.

Techniques: Infection, Blocking Assay, Expressing, Inhibition, Concentration Assay

Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet:

Article Snippet: Anti-human ACE2 (polyclonal goat IgG Poly5036) - AF647 (in-house conjugation) , Biolegend , Cat# 503602; RRID: AB_2892475.

Techniques: Conjugation Assay, Purification, Blocking Assay, Recombinant, Control, Virus, Saline, Modification, Staining, Library Quantification, Antibody Labeling, Flow Cytometry, Software, Cytometry, Sequencing

Journal: Oncotarget

Article Title: MEK inhibitors reduce cellular expression of ACE2, pERK, pRb while stimulating NK-mediated cytotoxicity and attenuating inflammatory cytokines relevant to SARS-CoV-2 infection

doi: 10.18632/oncotarget.27799

Figure Lengend Snippet: The effects of chloroquine, hydroxychloroquine, remdesivir and MEK inhibitors (VS-6766, selumetinib and trametinib) on ACE2, TMPRSS2 and IL-6 in human lung and colon cells are shown. ( A ) H1975 human NSCLC cells were treated with the indicated drugs and doses for 48 hours. Glycosylated ACE2, ACE2, TMPRSS2 (full-length and Serine Protease-domain), IL-6 were probed with the cell signaling 4355, Abnova PAB13444, Sigma MABF2158, and Sigma SAB1408591 antibodies. β-Actin was probed with Sigma A5441 as a loading control. ( B ) Effect of remdesivir and VS-6766 on ACE2(-1119)-Luc reporter and cell viability (bioluminescence images are shown in the grids on the left). HCT116 human colorectal cancer cells were transiently transfected with ACE2(-1119)-Luc reporter for 24 hours followed by remdesivir and VS-6766 treatment for 24 hours at the indicated doses. First D-Luciferin was added to acquire ACE2-Luc reporter bioluminescence images (left panel) with the Xenogen IVIS system. After the bioluminescence signal decayed, CellTiter-Glo was added to acquire cell viability images (right panel; “CTG”) with the IVIS system. The right bar graph shows ACE2 mRNA level in HCT116 cells treated with remdesivir and VS-6766 for 24 hours. mRNA levels were quantified by qRT-PCR. Data were normalized to GAPDH expression and plotted relative to cells treated with DMSO as a control. Data are expressed as mean ± SD. ( C ) Remdesivir alone increased ACE2 protein expression. H1299 human NSCLC cells, MSTO-211H human mesothelioma cells, and Calu-3 human NSCLC type II alveolar cells were treated with remdesivir and VS-6766 for 48 hours. ACE2 was probed with Abnova PAB13444 antibody, and β-Actin was probed with Sigma A5441 antibody as a loading control.

Article Snippet: To further investigate the correlation between MAPK-pERK activation and ACE2 expression we investigated the effects of remdesivir and VS-6766 on ACE2 expression and pERK expression in several human cell lines ( ).

Techniques: Transfection, Quantitative RT-PCR, Expressing

Journal: Oncotarget

Article Title: MEK inhibitors reduce cellular expression of ACE2, pERK, pRb while stimulating NK-mediated cytotoxicity and attenuating inflammatory cytokines relevant to SARS-CoV-2 infection

doi: 10.18632/oncotarget.27799

Figure Lengend Snippet: ( A ) Calu-3 human NSCLC type II alveolar cells were pretreated with 10 μM MEK inhibitor (VS-6766, trametinib or selumetinib) for 1 hr before incubation with 4.7 μM recombinant SARS-CoV-2 spike protein subunit 1 (S1, 0.118 μg/mL) or subunit 2 (S2, 0.275 μg/mL) for 2 hr. ACE2 protein detected with sc-390851 was increased following cell incubation with S1 and S2. All three MEK inhibitors suppressed increased ACE2. ( B ) HT-29 human colorectal cancer cells and BEAS-2B normal human bronchial epithelial cells were pretreated with 10 μM RAF/MEK inhibitor VS-6766 for 1 hr before incubation with 4.7 μM recombinant SARS-CoV-2 spike protein subunit 1 (S1, 0.118 μg/mL) and subunit 2 (S2, 0.275 μg/mL) for 2 hr. Recombinant SARS-CoV-2 spike protein subunits increased pERK expression which was completely abrogated by VS-6766 treatment.

Article Snippet: To further investigate the correlation between MAPK-pERK activation and ACE2 expression we investigated the effects of remdesivir and VS-6766 on ACE2 expression and pERK expression in several human cell lines ( ).

Techniques: Incubation, Recombinant, Expressing

Journal: Oncotarget

Article Title: MEK inhibitors reduce cellular expression of ACE2, pERK, pRb while stimulating NK-mediated cytotoxicity and attenuating inflammatory cytokines relevant to SARS-CoV-2 infection

doi: 10.18632/oncotarget.27799

Figure Lengend Snippet: ( A ) Effects of 5 μM remdesivir (RDV), RAF/MEK inhibitor VS-6766, or the combination on ACE2, ERK1/2, and pERK protein expression. HCT116 human colorectal cancer cells, H1975 human NSCLC cells and BEAS-2B normal human bronchial epithelial cells were treated with 5 μM VS-6766 or/and 5 μM Remdesivir for 48 hr. Remdesivir alone increased pERK protein expression in all three cell lines, which was completely depleted by VS-6766 treatment. Effects of 5 μM RDV, VS-6766, or the combination on ACE2, ERK1/2, and pERK protein expression in ( B ) normal human lung cells and ( C – E ) human lung cancer cells are shown. Serum-deprived cells were plated and cultured in medium containing 1% FBS for the indicated amount of time. For serum-stimulated cells, FBS was added to a final concentration of 10% for 24 hours (B–E, left 2 panels). Cells were plated in 10% serum for 16 hours, then media was removed and replaced with 1% FBS for serum-deprived cells. For serum-stimulated cells, FBS was added to a final concentration of 10% for 4 hours (E, right panel). ACE2 (PAB), ACE2 (CS), ERK1/2, and pERK were probed with Abnova PAB13444, Cell Signaling 4355, Cell Signaling 9102, and Cell Signaling 4370 antibodies. Ran was probed with BD Biosciences 610341 antibody as a loading control. ( F ) Modulation of pRb and ACE2 expression in H1299 cells with serum starvation, stimulation, and MEKi treatment. Control cells were grown with the normal 10% serum throughout the treatment. Serum starved cells were plated in 10% serum and incubated for 16 hours, then grown in media containing 0% serum for 72 hours. Serum stimulated cells were similarly starved for 72 hours, then stimulated with media containing 20% serum for 48 hours. All drug treated cells received 5 mM RDV, VS-6766, or the combination 48 hours before harvesting. ( G ) Modulation of ACE2 expression with serum starvation and stimulation. Four different cell lines (2 lung, 1 colorectal, and 1 breast cancer) were grown in 10% serum for 16 hours then were starved in 0% FBS for 0 (no starvation), 24, 48, or 72 hours. Cells were stimulated with 20% FBS and harvested either 0 (no stimulation), 12, or 24 hours later. An increase in pRb relative to total Rb was seen upon stimulation and this correlated with ACE2 levels. pERK correlation with ACE2 was heterogeneous, with a correlation seen in MCF7 cells but not H1299 or HCT-116 cells (three left-most panels). pRb relative to total Rb decreased upon starvation and increased upon stimulation, which correlated with ACE2 expression (H460 and HCT-116 in right-most panels).

Article Snippet: To further investigate the correlation between MAPK-pERK activation and ACE2 expression we investigated the effects of remdesivir and VS-6766 on ACE2 expression and pERK expression in several human cell lines ( ).

Techniques: Expressing, Cell Culture, Concentration Assay, Incubation