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: JCI Insight

Article Title: IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function

doi: 10.1172/jci.insight.158362

Figure Lengend Snippet: Anti-ACE2 IgM antibodies were assayed by ELISA in serum from 66 patients with COVID-19, 52 COVID-19 patients with multiple bleeds available, 133 disease controls, and 30 healthy controls. The functional consequences of these antibodies were investigated with 3 different assays using IgM purified from anti-ACE2 IgM–positive sera. MDA5 DM, melanoma differentiation-associated 5 dermatomyositis.

Article Snippet: The anti-ACE2 ELISA was validated by blotting purified recombinant human ACE2 and using a second source of recombinant human ACE2 (SinoBiological 10108-H08H).

Techniques: Enzyme-linked Immunosorbent Assay, Functional Assay, Purification

Journal: JCI Insight

Article Title: IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function

doi: 10.1172/jci.insight.158362

Figure Lengend Snippet: ( A – C ) Antibodies were assayed by ELISA in the combined COVID-19 cohort ( n = 118 patients). ( A ) Number of patients with and without anti-ACE2 IgM antibodies shown grouped by disease severity: 27.2% of severe patients were anti-ACE2 positive compared with 3.8% with moderate COVID-19 ( P = 0.0009; 2-tailed Fisher’s exact test). ( B and C ) Data from anti-ACE2 IgM ( B ) and IgG ( C ) ELISAs are presented as corrected OD 450 absorbance units. These data were obtained on all the patients with COVID-19 in A , as well as from 30 healthy controls. Red dots in the IgG panel denote IgG-positive samples that also have anti-ACE2 IgM antibodies. The horizontal line on each plot represents the cutoff for assigning a positive antibody status. ( D ) Anti-ACE2 IgM antibodies are detected in patients with COVID-19 but not in other infectious and autoimmune disease controls.

Article Snippet: The anti-ACE2 ELISA was validated by blotting purified recombinant human ACE2 and using a second source of recombinant human ACE2 (SinoBiological 10108-H08H).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function

doi: 10.1172/jci.insight.158362

Figure Lengend Snippet: ( A ) Age, ( B ) BMI, ( C ) sex, and levels of ( D ) temperature, ( E ) CRP, ( F ) D-dimer, and ( G ) neutrophils were compared between the anti-ACE2 IgM–positive and –negative COVID-19 patient groups. Red and blue colors denote anti-ACE2 IgM antibody–positive and –negative status, respectively. Box plots show median, 25th and 75th percentiles, and whiskers min to max. ( D – G ) Anti-ACE2 IgM-positive patients had higher average body temperature beginning early after hospital admission, followed by elevated CRP and D-dimer measurements. The IgM anti-ACE2–positive group had statistically significantly higher average temperatures, CRP, and D-dimer levels over the first 10 days of hospitalization than the IgM-negative group ( P = 0.0001, 0.02, and 0.001, respectively). Average absolute neutrophil levels ( G ) were not statistically different between the 2 groups. Analyses in panels D – G use linear mixed effects model Wald test with 4 degrees of freedom (see Statistics ). ( H ) Longitudinal analysis of anti-ACE2 IgM antibodies. For all those anti-ACE2 IgM–positive patients with multiple banked sera available (16/18), anti-ACE2 IgM and IgG antibodies were quantitated over time. Red and blue lines on each plot denote anti-ACE2 IgM and IgG antibodies, respectively. Solid black bars represent steroid treatment periods. Additional examples are shown in .

Article Snippet: The anti-ACE2 ELISA was validated by blotting purified recombinant human ACE2 and using a second source of recombinant human ACE2 (SinoBiological 10108-H08H).

Techniques:

Journal: JCI Insight

Article Title: IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function

doi: 10.1172/jci.insight.158362

Figure Lengend Snippet: Equilibrium binding titrations ( A – C ) and kinetic traces ( D – K ) of immobilized ACE2 and IgM purified from human serum, as measured by biolayer interferometry. ( A – C ) Normalized responses at the indicated concentrations of purified IgM from 3 healthy donors and 5 SARS-CoV-2–infected patients are plotted (data from 3 independent experiments). Equilibrium dissociation constants ( K D ) calculated by fitting to a 4-parameter logistic regression model are provided. ( D – K ) The indicated percentages (color-coded on the right side of K ) represent 2-fold dilutions of the 8 purified IgM preparations shown in A – C . Binding constants were determined via global fitting for each IgM sample using Octet Data Analysis software (assuming a 1:1 binding model). Quantitation of the data shown in A – K is provided in .

Article Snippet: The anti-ACE2 ELISA was validated by blotting purified recombinant human ACE2 and using a second source of recombinant human ACE2 (SinoBiological 10108-H08H).

Techniques: Binding Assay, Purification, Infection, Software, Quantitation Assay

Journal: JCI Insight

Article Title: IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function

doi: 10.1172/jci.insight.158362

Figure Lengend Snippet: ( A ) Anti-ACE2 IgM antibodies from patient CV-1 or control do not inhibit ACE2 activity. Positive and negative controls were ACE2 alone, and ACE2 plus ACE2 inhibitor, respectively (see ). ( B ) IgM antibodies to ACE2 activate complement. Purified IgMs from anti-ACE2 IgM–positive COVID-19 patients ( n = 8) and healthy controls ( n = 11) was used for C1q binding assays. Values are means from 2 independent experiments performed on different days. *** P < 0.0001, Mann-Whitney test. (C–G) Anti-ACE2 IgM affects the pulmonary endothelium. ( C ) Phase image of a pulmonary microvessel (top). Fluorescence images of microvessels exposed to anti-ACE2–negative IgM (HC) or anti-ACE2–positive IgM (CV) after perfusion with 10 kDa dextran (lower). Representative images across n = 3 to 6 independent experiments for each IgM condition are shown. ( D ) ACE2 and CD31 microvessel staining following 24-hour perfusion with IFN-α/γ. Representative images across n = 3 independent experiments are shown. ( E ) C3c staining after perfusion with IFN and anti-ACE2–positive or control IgM (3.33 μg/mL). Representative images across n = 3 independent experiments are shown. ( F ) Permeability of microvessels perfused with IFN and anti-ACE2–positive IgM (CV) or anti-ACE2 negative IgM (HC) (100 μg/mL). D.L., detection limit. A linear mixed effects model was used to test the effect of anti-ACE2–positive IgM on permeability. n = 3 to 5 for each IgM condition; each dot represents an independent replicate. ( G ) Inhibition of microvessel permeability to 10 kDa dextran in response to anti-ACE2 IgM is IFN dependent. Low CV-1: 3.33 μg/mL; affinity-purified anti-ACE2 IgM: 100 ng/mL. To compare each condition to control, a Kruskal-Wallis test followed by Dunn’s multiple-comparison test was performed. n = 4 to 6 for each IgM condition; each dot represents an independent replicate.

Article Snippet: The anti-ACE2 ELISA was validated by blotting purified recombinant human ACE2 and using a second source of recombinant human ACE2 (SinoBiological 10108-H08H).

Techniques: Activity Assay, Purification, Binding Assay, MANN-WHITNEY, Fluorescence, Staining, Permeability, Inhibition, Affinity Purification

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: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: High-throughput of ReFRAME, Pathogen Box, TargetMol and Cathepsin L drug libraries for SARS-CoV-2 antiviral compounds. A. Schematic of the spike protein of SARS-CoV-2. RBD: receptor binding domain. B. Schematic of the High-throughput assay. Compounds were pre-spotted in 1536-well plates. Next, 2000 HEK293T-ACE2 cells were added to each well and pre-incubated with each compound for 1 h, followed by infection with MLV reporter luciferase virus pseudotyped with the SARS-CoV-2 Spike protein (SARS2-S) or VSV-G protein (VSV-G). Luciferase was measured 48 h later. C. Summary of the ReFRAME library results. Conc.: concentration. D. Distribution of Z-Score for primary screens of each library. Scatter plot of Z_Score for all samples tested from the ReFrame library ( N = 1; circle) and other libraries ( N = 3; Cathepsin L: square; Pathogen Box: cross; TargetMol: filled circle). Total of 16,320 samples. Positive controls: orange; Negative control: cyan; Hit compounds: red; non-hit compounds: black. E. Summary of the 3 other libraries results. F. ReFrame library screening against different targets: SARS2-S, 3CLpro and PLpro. Venn diagram analysis of comparison between hits from SARS2-S entry, 3CLpro and PLpro assay against ReFRAME library results. There are 419 compounds that are SARS2-entry specific potential inhibitors. G. Robustness in terms of Z’ score of each screen for each library.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: High Throughput Screening Assay, Binding Assay, Incubation, Infection, Luciferase, Virus, Concentration Assay, Negative Control, Library Screening, Comparison

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Summary of the selected Cathepsin L, Pathogen box and TargetMol compounds in this study. Activity of the selected compounds against the different MLV pseudotyped viruses in HEK293-ACE2 cells and their respective cytotoxicity. Values for SARS2-S, VSV-G and toxicity are mean ± SEM of 2–4 independent experiments. TI: therapeutic index. * n = 1.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Targets of the selected compounds and SARS-CoV-2 wild type infection. A. Description of the targets of the different hits from all the studied libraries. B. Antiviral activity of the 2 best hits in the SARS-CoV-2-induced CPE assay. Vero E6 cells treated with test compounds for two hours were infected with SARS-CoV-2 at an MOI of 0.05, then incubated for three days in the presence of compound. Cell viability (protection from virus-induced CPE) was measured with CellTiter-Glo. C and D. Antiviral effect was measured with a subset of Vero E6 cells expressing a low (C) and high (D) level of ACE2. E. Cytotoxicity of selected compounds in Vero E6 cells. Cytotoxicity was tested in the same conditions with cell culture media instead of the virus. F. Virus yield reduction activity of selected compounds. Vero E6 cells infected with SARS-CoV-2 at an MOI of 0.05 were cultured in the presence of test compound (5 µM) and the supernatant was harvested after 24 and 48 h of incubation. The Progeny virus was enumerated with a plaque assay using an Avicel overlay in fresh Vero E6 cells. N = 3 experiments were performed for infectivity assays and n = 2 for the cytotoxicity assays. **** P < 0.0001, Two-way ANOVA with Dunnett's multiple comparisons test against DMSO control.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Infection, Activity Assay, Incubation, Virus, Expressing, Cell Culture, Plaque Assay, Control

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: SR-914 “calpeptin” specifically blocks SARS-CoV entry. A. Its activity against SARS2-S in HEK293T-ACE2-TMPRSS2 cells. Cells were incubated with different concentrations of drugs, then infected with SARS2-S or VSV-G. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 2 to 4 independent experiments. B. Time of drug addition experiment schematic. Infection was performed for 1 h with or without drugs, Vero CCL81 cells were then washed, and fresh media was added with or without drugs. C. Time of drug addition experiment result. SR-914 was used at 10 µM. E64d at 20 µM. Calp.: calpeptin = SR-914. NI: not infected. Shown is the mean ± SEM of 4 to 6 independent experiments. D. Luciferase complementation assay schematic. The reporter consists of a split Firefly luciferase protein connected by a cleavable peptide for the tested protease. Upon cleavage of the peptide, the luciferase protein undergoes dimerization for an active state. DnaE intein helps in this dimerization. E. Its activity against SARS2-S Entry, 3CLpro and PLpro. C-: negative control. C+; positive control. Shown is the mean ± SD of 3 independent experiments. One-way ANOVA followed by Tukey's post-test were used for statistical comparisons. *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay, Incubation, Infection, Luciferase, Negative Control, Positive Control

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Breath of activity of calpeptin against various SARS-CoVs. A. Its activity against SARS1-S in HEK293T-ACE2 cells. HEK293T-ACE2 cells were incubated with different concentrations of calpeptin, then infected with SARS1-S. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 2 independent experiments. B. Schematic of the substituted residues in the S protein of the highest threat of SARS-CoV-2 strains. C. Evolution of the S protein residues at the position 417, 484, 501 and 614 from 2019 to February 2021. Modified figure from https://nextstrain. org/ncov/global?branchLabel=none& c =gt-S_417,484,501,614& l =clock. D. Activity of the new emergent variants. HEK293T-ACE2 cells were infected with different mutants of SARS2-S. The day after, a medium change was performed. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 3 independent experiments. WT: wild type, SA: South Africa, UK: United Kingdom. E. Activity of calpeptin activity against crucial mutations present in the S protein of the new emergent strains. Similar experiment than D but calpeptin was added during infection and after medium change. Shown is the mean ± SEM of n = 2–5 independent experiments. Two-way ANOVA followed by Dunnett's post-test were used for statistical comparisons. *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay, Incubation, Infection, Luciferase, Modification

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: Dysregulated expression of primary cilium-associated genes in SARS-CoV-2 infected patient lung samples. A The Venn diagram presents genome-wide RNA sequencing results from patient lung samples, revealing differentially expressed genes (DEGs) associated with primary cilium. The DEGs were defined by log2 fold change > 1.5, with adjusted P < 0.05. P values were adjusted using the Benjamini–Hochberg correction. The analysis showing 3,972 DEGs in patient samples and 956 primary cilia-related genes in CiliaCarta, with 129 overlapping genes functionally linked to primary cilium. B The list of 129 ciliary DEGs identified in the SARS-CoV-2 patient lung samples. Gene ranked by the differently expressed level. Blue dots represent down-regulated genes, red dots represent up-regulated genes. Colour gradient and size of dots correlate with the degree of fold change. C Volcano plots of 129 DEGs in the patient lung samples overlap with the genes associated with ciliary functions. DEGs at -log10 > 2 and |log2 fold change|> 2 were indicated. (adjusted p values < 0.05, P values adjusted using the Benjamini–Hochberg correction) 61 up-regulated genes presented as red dots, 68 down-regulated genes presented as blue dots. Top 15 and two prominent ciliary genes ARL13B and ADCY3 are indicated. D Gene Ontology (GO) enrichment analysis for Biological Processes among ciliary DEGs from SARS-CoV-2 patient lung samples. Terms are ranked by their enrichment score, calculated using Gene Set Enrichment Analysis (GSEA). E Gene Set Enrichment Analysis (GSEA) reveals key signaling pathways associated with ciliary DEGs from SARS-CoV-2 patient lungs, ranked by enrichment score. F Ciliary DEGs from SARS-CoV-2 patient lung samples grouped by ciliary functional category. G Venn diagram and table show overlap between ACE2 interactors from BioGRID (979 proteins) and ciliary genes from CiliaCarta (956 genes) and Syscilia (303 genes). 56 and 14 overlapping genes were identified in each cilia database, respectively. Two well-studied ciliary components, namely ARL13B and TULP3 are identified in the intersections (highlighted yellow). H Gene Ontology (GO) enrichment analysis for Biological Processes among ciliary ACE2 interactors. Terms are ranked by their enrichment score, calculated using Gene Set Enrichment Analysis (GSEA)

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Expressing, Infection, Genome Wide, RNA Sequencing, Protein-Protein interactions, Functional Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: TULP3 colocalizes with ACE2 on the primary cilium axoneme and physically interacts with ACE2. A Representative immunofluorescence images show the accumulation and co-localization of TULP3 and ACE2 on the primary cilium of A549 cells. B Histogram illustrates co-localization profile of endogenous ACE2 and TULP3 expressions on ARL13B-positive cilia axoneme of A549. C Mander’s Coefficients analysis of ACE2 and TULP3 co-localization in cytoplasm versus primary cilia of A549 cell. The data represents quantifications from three independent experiments, with 10–20 data points measured in each experiment. Statistical analysis is performed using Paired Student’s t-Test, Two tailed. P values: ** < 0.01, *** < 0.001, **** < 0.0001. D Representative Western-blot images illustrate the co-IP experiment of ACE2 and TULP3 on ACE2-GFP overexpressing HEK293T cell line. IP: immunoprecipitation; IB: immunoblot. E Graph depicts densitometry analysis of TULP3 signal intensity in ACE2 and IgG immunoprecipitation groups. The data represents quantifications from three independent experiments. Statistical analysis is performed using Unpaired Student’s t-Test, Two tailed. P value * < 0.05. F Graph depicts densitometry analysis of ACE2 signal intensity in TULP3 and IgG immunoprecipitation groups. The data represents quantifications from three independent experiments. Statistical analysis is performed using Unpaired Student’s t-Test, Two tailed. P value * < 0.05. G Representative Western-blot images illustrate the co-IP experiment of ACE2 and ARL13B on ACE2-GFP overexpressing HEK293T cell line. IP: immunoprecipitation; IB: immunoblot. H TULP3–ACE2 interaction interface predicted from AlphaFold3 . TULP3 (light blue, residues 271–430) and ACE2 (light red, residues 749–773) are shown with key interface residues highlighted as sticks (blue for TULP3, red for ACE2). The TULP3 cargo-binding face (β8–β12, highlighted in marine) engages the membrane-proximal ACE2 segment. Yellow dashed lines represent top recurring contacts across models. ACE2 residue I761, involved in all major contacts, is labelled

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Two Tailed Test, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Binding Assay, Membrane, Residue

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: TULP3 regulates ACE2 localization to the primary cilium. A Representative immunofluorescence images showing reduced ACE2 expression on the primary cilium in the shControl and TULP3 KD groups. B Graphs showing reduced ACE2 enrichment ratio in the (top) TULP3 and (bottom) ARL13B KD groups. ACE2 enrichment ratio was calculated as (ACE2 intensity per area on primary cilium)/(ACE2 intensity per area on whole cell). The data represents quantifications from three to four independent experiments, n = 14–17 measurements in each experiment. Statistical analysis is performed using (top) One-way ANOVA followed by Dunnett’s multiple comparisons and (bottom) Unpaired Student’s t-test. P values: * < 0.05, *** < 0.001, ns = not significant. C Representative immunofluorescence images show ciliary ACE2 expression on the primary cilium in TULP3 WT and TULP3 mut12 overexpression groups (pseudo-color magenta). D Graph shows ciliary ACE2 enrichment ratio in the TULP3 WT rescue and TULP3. mut12 overexpression groups. ACE2 enrichment ratio was calculated as (ACE2 intensity per area on primary cilium)/(ACE2 intensity per area on whole cell). The data represents quantifications from four independent experiments, n = 10–20 measurements in each experiment. Statistical analysis is performed using One-way ANOVA followed by Tukey’s multiple comparisons test. P values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Expressing, Over Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: Schematic diagram illustrating our proposed model. TULP3 functions as a ciliary adaptor protein that governs the targeting of ACE2 to the ciliary axoneme, acting partially through anchoring to the IFT-A transport complex

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: Attachment and accumulation of the viral spike protein on the ACE2-enriched primary cilium. A Representative immunofluorescence images show the negative control without exposure to spike protein. A ” Histogram illustrating the ciliary co-localization and expression profiles on of spike and ACE2 signals the negative control. B Representative immunofluorescence images show the accumulation and co-localization of viral recombinant spike protein on endogenously expressed ACE2 on the ARL13B. + primary cilium. B ” Histogram illustrating the co-localization and expression profile of three fluorescence channels reflecting viral spike protein, ARL13B and ACE2 respectively, on the primary cilium. C Graph depicts the spike expression intensity on ACE2-negative and ACE2-positive primary cilia. ACE2 pixel intensities per cilia area greater than 500 were defined as ACE2-positive primary cilia, whereas below 500 were counted as ACE2-negative primary cilia. The data represent quantifications from three independent experiments, with n = 10–20 for each group in each experiment. P values: ** < 0.01

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Negative Control, Expressing, Recombinant, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: Host cell entry factors for SARS-CoV-2 are endogenously expressed and enriched in primary cilia across multiple SARS-CoV-2 susceptible cell types. A Representative immunofluorescence images show the endogenous expression of ACE2 (green) on the primary cilium (red) of human lung carcinoma, A549, human retinal pigment epithelial cells (hTERT-RPE1), as well as neuronal cell types namely neuroblastoma, SH-SY5Y, and human induced-pluripotent stem cell derived neural progenitor cells (hiPSC-NPCs) respectively. B The violin plots depict the quantification of the per area expression intensity of ACE2 in the primary cilium in relation to the entire cell. The data represents quantifications from three independent experiments. Statistical analysis is performed using Wilcoxon matched-pairs signed rank test. P values: ** < 0.01,**** < 0.0001. ~ 10 to 25 cells were analysed in each group of three independent experiments. C The violin plot depicts the percentage of ACE2-expressing primary cilia across different human cell types that are susceptible to SARS-CoV-2 infection. Data illustrates three to eight independent experiments for each group. Statistical analysis is performed using Kruskal–Wallis Test and Dunn’s multiple comparisons test. P values: * < 0.05, ** < 0.01. D Representative immunofluorescence images showing the endogenous expression of NRP1 (green) on the primary cilium (red) of human lung carcinoma, A549 and hiPSC-NPCs

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Expressing, Derivative Assay, Infection

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: Genetic perturbation of primary cilia formation reduces SARS-CoV-2 infection in human host cells. A - B Representative Western blot images and the corresponding graphs show the knockdown efficiency of sh ARL13B and sh IFT88 in the ( A ) A549 and ( B ) RPE1 knockdown cells. Knockdown efficiency was quantified by normalizing the signal intensity of the target protein from each knockdown sample to its respective control group. The data represents quantifications from three independent experiments. Statistical analysis is performed using One-way ANOVA followed by Dunnett’s multiple comparisons test. P values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. ns = not significant. Error bar: SD. C Representative immunofluorescence images show a reduced number of ciliated cells in the ARL13B KD and IFT88 KD groups in A549 (top panel) and RPE1 (bottom panel) cell lines. D - E Graphs depict the percentage of primary cilia-bearing (ciliated) cells in the ARL13B KD and IFT88 KD groups in ( D ) A549 and ( E ) RPE1 respectively. The data represents quantifications from five independent experiments. Statistical analysis is performed using One-way ANOVA followed by Dunnett’s multiple comparisons test. P values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. ns = not significant. F - G Graphs depict flow cytometry analysis of the relative infection rate of different SARS-CoV-2-GFP pseudovirus variants upon perturbation of primary cilia in the ARL13B - and IFT88- knockdown human lung and retinal cells respectively. The percentage of infected cells was determined by flow cytometry analysis of GFP-positive infected cells. The data represents quantifications from three independent experiments. Each dot represents the mean value of an independent experiment. Statistical analysis is performed using Two-way ANOVA, Tukey's multiple comparisons test. P values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. ns = not significant. Error bar: S.E.M. H Schematic diagram summarizes the findings, showing that the loss of ACE2-enriched cilia decreases SARS-CoV-2 viral uptake. Created with https://BioRender.com

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Infection, Western Blot, Knockdown, Control, Immunofluorescence, Flow Cytometry