Journal: mBio

Article Title: SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein

doi: 10.1128/mbio.00820-23

Figure Lengend Snippet: SCV2 pseudovirus induces SCV2-specified gene expression in NHBE. NHBE cells were infected with SCV2 spike or control pseudovirus for 72 h, and ( A ) the infection efficiency was imaged by DsRed fluorescent signal (32 ×, scale bars of 100 µm). Gene expression of ( B ) HES1 and ( C ) KLF2 was examined by qPCR and normalized by housekeeping gene GAPDH. The error bars represent ±standard error of the mean (SEM) ( n = 3). Statistics were determined by Welch’s test, with P < 0.05 considered statistically significant. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HES1, hes family BHLH transcription factor 1; KLF2, KLF transcription factor 2; NHBE, normal human bronchial epithelial; Spike, SARS-CoV-2 spike glycoprotein; VSV-G, VSV glycoprotein.

Article Snippet: Full-length VSV glycoprotein (VSV-G) (Addgene, #12259) and pRP-Neo-EF1A-SARS-Cov2-Spike delta 18, fused with a DsRed tag, were kindly gifted from Vasudevan, Anand’s lab. Plasmid for the SARS-CoV-2 spike delta 18 glycoprotein has a C-terminal deletion of the last 18 amino acids to improve binding to ACE2.

Techniques: Gene Expression, Infection, Control

Journal: mBio

Article Title: SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein

doi: 10.1128/mbio.00820-23

Figure Lengend Snippet: SCV2 infection enhances metabolism in the host cells. Using KEGG pathway analysis, differentially expressed genes involved in ( A ) nucleotide, ( B ) amino acid, ( C ) fatty acid, and carbohydrate metabolisms were shown in IAV/mock and SCV2/mock at 24 hpi. After 72 h of pseudovirus infection in NHBE cells, gene expression of ( E ) glycolysis rate limiting PKLR, ( F ) SIRT3, and ( G ) mitochondrial marker TOMM22 was measured by qPCR and normalized by housekeeping gene GAPDH (5–9 inserts from two donors), and ( H ) the mitochondrial localization was observed by TOMM22 immunofluorescent staining. The immunofluorescence images were taken at 63× oil objective, scale bars of 20 µm. The error bars represent ±standard error of the mean (SEM) (two donors). Statistics were determined by Welch’s test, with P < 0.05 considered statistically significant. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. PKLR, pyruvate kinase L/R; SIRT3, NAD+-dependent protein deacetylases 3; TOMM22, translocase of the outer membrane of mitochondria; Spike, SARS-CoV-2 spike glycoprotein; VSV-G, VSV glycoprotein.

Article Snippet: Full-length VSV glycoprotein (VSV-G) (Addgene, #12259) and pRP-Neo-EF1A-SARS-Cov2-Spike delta 18, fused with a DsRed tag, were kindly gifted from Vasudevan, Anand’s lab. Plasmid for the SARS-CoV-2 spike delta 18 glycoprotein has a C-terminal deletion of the last 18 amino acids to improve binding to ACE2.

Techniques: Infection, Gene Expression, Marker, Staining, Immunofluorescence, Membrane

Journal: mBio

Article Title: SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein

doi: 10.1128/mbio.00820-23

Figure Lengend Snippet: SCV2 spike protein infection impairs metabolism in human epithelial cells. ( A ) The pseudovirus infection efficiency of Calu3 cells after 48 h post infection was examined by DsRed fluorescent signal (32×, scale bars of 100 µm). ( B ) ECAR and ( C ) OCR were examined in the pseudovirus-infected Calu3 cells. ( D ) Basal respiration, ( E ) maximal respiration, and ( F ) ATP-linked respiration were shown. Gene expression of ( G ) SIRT3 and ( H ) TOMM22 as well as ( I ) immunofluorescence of TOMM22 (green) and Hoechst (blue) staining were taken at 63× oil objective, scale bar of 20 µm. The error bars represent ±standard error of the mean (SEM) (4–10 inserts/group). Statistics were determined by Welch’s test, with P < 0.05 considered statistically significant. ECAR, extracellular acidification rate; OCR, oxygen consumption rate; RHOB, ras homolog family member B; SIRT3, NAD+-dependent protein deacetylases 3; TOMM22, translocase of the outer membrane of mitochondria; Spike, SARS-CoV-2 spike glycoprotein; VSV-G, VSV glycoprotein.

Article Snippet: Full-length VSV glycoprotein (VSV-G) (Addgene, #12259) and pRP-Neo-EF1A-SARS-Cov2-Spike delta 18, fused with a DsRed tag, were kindly gifted from Vasudevan, Anand’s lab. Plasmid for the SARS-CoV-2 spike delta 18 glycoprotein has a C-terminal deletion of the last 18 amino acids to improve binding to ACE2.

Techniques: Infection, Gene Expression, Immunofluorescence, Staining, Membrane

Journal: mBio

Article Title: SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein

doi: 10.1128/mbio.00820-23

Figure Lengend Snippet: SCV2 infection disrupts cell-cell adhesion in the human epithelium. Using KEGG pathway analysis, differential expression of genes involved in ( A ) tight junction, ( B ) cell adhesion, ( C ) focal adhesion, and ( D ) regulation of actin cytoskeleton was shown in IAV/mock and SCV2/mock at 24 hpi. After 72 h of the pseudovirus infection in NHBE cells, expressions of ( E ) CFL1 transcript, ( F ) CFL1 protein, and ( G ) RHOB transcript were examined ( n = 3–12). ( G ) Immunofluorescence of TOMM22 (green) and Hoechst (blue) staining was taken at 63× oil objective, scale bar of 10 µm. TEER was measured in the pseudovirus-infected ( H ) NHBE cells and ( I ) Calu3 cells ( n = 8). The error bars represent ±standard error of the mean (SEM). Statistics were determined by Welch’s test, with P < 0.05 considered statistically significant. CFL1, cofilin-1; ECAR, extracellular acidification rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OCR, oxygen consumption rate; TEER, transepithelial electrical resistance; TOMM22, translocase of the outer membrane of mitochondria; Spike, SARS-CoV-2 spike glycoprotein; VSV-G, VSV glycoprotein.

Article Snippet: Full-length VSV glycoprotein (VSV-G) (Addgene, #12259) and pRP-Neo-EF1A-SARS-Cov2-Spike delta 18, fused with a DsRed tag, were kindly gifted from Vasudevan, Anand’s lab. Plasmid for the SARS-CoV-2 spike delta 18 glycoprotein has a C-terminal deletion of the last 18 amino acids to improve binding to ACE2.

Techniques: Infection, Quantitative Proteomics, Immunofluorescence, Staining, Membrane

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative double immunofluorescence staining of ACE2 and endothelial cell marker CD31 in the blood vessels of human nasal turbinates using six different anti-ACE2 antibodies and anti-CD31. b Double immunofluorescence staining of ACE2 and type II pneumocyte marker mucin 1 (MUC1) in the human lung using six different anti-ACE2 antibodies and anti-MUC1. Abcam ab15348 clone yielded the most robust staining of pneumocytes, while the other clones showed negligible or less specific membrane staining. Scale bars: 20 μm (top) and 5 μm (bottom).

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Double Immunofluorescence Staining, Marker, Staining, Clone Assay, Membrane

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: Representative images of human tissues on a tissue microarray (TMA) stained by chromogenic immunohistochemistry using antibodies targeting the ACE2 protein (brown) and counterstained with hematoxylin (blue). Highest ACE2 expression was observed in the villi of the intestinal tract (jejunum), renal tubules, testis, and glandular cells in the seminal vesicle. Minimal to no/non-specific staining can be seen in the heart, stomach, spleen, skin, and liver. Staining of lung pneumocytes was observed using Abcam ab15348, and less specifically with Sigma HPA000288 (Fig. 2b; Supplementary Table 1 ). Scale bars: 100 μm.

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Microarray, Staining, Immunohistochemistry, Expressing

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative double immunofluorescence staining of ACE2 and acetylated α-tubulin (ACTUB) on normal human nasal turbinate, ethmoid sinus, uncinate process (sinus), trachea, and bronchus, using anti-ACE2 and anti-ACTUB antibodies, respectively. b Representative double immunofluorescence staining of ACE2 and ACTUB on normal C57BL/6J mouse nasal turbinate and trachea. c Immunofluorescent staining of (top panel) ACE2, cilia marker ADP-ribosylation factor-like protein 13B (ARL13B), and cilia centrosome marker FGFR1 oncogene partner (FOP); (bottom panel) ACE2, and cilia markers ACTUB and ARL13B in a ciliated mouse cell line, IMCD3. d Immunofluorescent staining of ACE2 in the primary cilia of IMCD3 cells transiently transfected with human ACE2 (yellow outline) compared to endogenous mouse ACE2 (blue outline). e Quantified percentages of endogenous ACE2-positive cilia (34.67 ± 13.58%; control (Ctrl)) versus cilia with overexpressed human ACE2 (82.67 ± 4.73%). Ciliated cells were identified by staining of ARL13B. Error bars represent mean ± SD. ( n = 100 cells examined per experiment over three independent experiments). (Two-tailed Student’s t test, ** p = 0.004). f Representative multiplexed images of in situ hybridization against the SARS-CoV-2 Spike mRNA, in combination with immunofluorescence staining of ACE2 and the differentiated epithelial cell marker cytokeratin 8 (KRT8). SARS-CoV-2 Spike mRNA expression (red) was detected within ciliated epithelial cells containing motile cilia positive for ACE2 (green). The nuclei were stained using DAPI (blue) as a counterstain. Scale bars: 20 μm ( a , b top panels; f large panels); 5 μm ( a , b bottom panels; f small panels); 2 μm ( c , d ).

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Double Immunofluorescence Staining, Staining, Marker, Transfection, Control, Two Tailed Test, In Situ Hybridization, Immunofluorescence, Expressing

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative immunofluorescence double staining of ACE2 and mucin 5AC (MUC5AC) reveals absence of co-localization of ACE2 within secretory goblet cells in the human nasal turbinate, uncinate process, and bronchus. b Representative in situ hybridization using an ACE2 probe in combination with an anti-MUC5AC antibody. ACE2 mRNA expression (red dots) was not detected within goblet cells marked by MUC5AC in the nasal turbinate, uncinate process, and trachea. Nuclei were stained using DAPI. Scale bars: 20 μm (top) and 5 μm (bottom).

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Immunofluorescence, Double Staining, In Situ Hybridization, Expressing, Staining

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a No statistically significant changes in ACE2 expression was detected among patients less than or greater than 65 years of age, males versus females, and patients with varying smoking history. (Two-tailed Mann–Whitney test or Kruskal–Wallis test, p > 0.05). b No statistically significant difference in ACE2 expression was observed between healthy controls and patients with chronic rhinosinusitis with polyps (CRSwNP) or without polyps (CRSsNP). (Kruskal–Wallis test, p > 0.05). c No statistically significant difference in ACE2 expression was noted between distinct human nasal tissue sites/regions. (Two-tailed Mann–Whitney test or Kruskal–Wallis test, p > 0.05). UNC uncinate process, Turb nasal turbinates, Eth ethmoid sinus, NP benign nasal polyps. The bottom and top of the box plots represent the 25th and 75th percentiles, respectively. The bands within the box show the median value, and the whiskers extending from both ends of the boxes are minimum and maximum values. Each dot represents one patient.

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Quantification of ACE2 in controls and patients taking ARBs and ACEI. In the Stanford cohort, ACE2 is slightly but statistically significantly lower in patients taking ACEI (0.19 ± 0.02) compared to controls (0.26 ± 0.06). (Kruskal–Wallis test p = 0.021; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.043). There were no statistically significant differences in ACE2 expression between patients taking ARBs and controls in the Stanford, National Taiwan University Hospital (NTUH), and China Medical University Hospital (CMUH) cohorts. b In the Stanford cohort, when including only controls with hypertension (HTN) on other medications (“HTN w/o ARBs/ACEI”), ACE2 expression was statistically different between the groups (Kruskal–Wallis test, p = 0.044) but Dunn’s multiple comparison post-hoc test did not reveal any statistical significance between the three groups. No statistically significant differences were seen among patients taking ARBs compared to controls. c When cohorts from all three institutions were normalized by Z -score and integrated, patients taking ACEI (−0.72 ± 0.42) had a lower ACE2 expression compared to controls with hypertension (0.41 ± 1.07). (Kruskal–Wallis test, p = 0.032; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.043). Patients taking ARBs (−0.15 ± 0.95) showed a trend towards lower ACE2 compared to controls with hypertension, but this was not statistically significant. d ACE2 expression among patients of older (≥65 years) and younger (<65 years) age taking ARBs or ACEI was not statistically divergent from control patients of the same age group. (Kruskal–Wallis test, p > 0.05). e ACE2 expression among male and female patients on ARBs or ACEI trended comparably or lower than same-sex controls except for males taking ARBs in the CMUH group who showed a trend towards higher ACE2 expression. No statistically significant differences were observed. (Kruskal–Wallis test, p > 0.05). f Among non-smokers, there was a statistically significant trend towards lower ACE2 expression in patients taking ACEI compared to controls in the Stanford group (Kruskal–Wallis test, p = 0.021; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.035). No statistical significance was observed with the non-smokers on ARBs. All data are noted as mean ± SD. Kruskal–Wallis test was used for three group comparisons and two-tailed Mann–Whitney test was used for two-group comparisons. Box plots are similar in format to Fig. .

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: Comparison, Expressing, Medications, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: The luminal differentiated airway epithelial cells consist of ciliated columnar cells (~80%) and secretory goblet cells (~20%). Club cells are infrequently found in the human upper airway. The basal cell layer, which faces the lamina propria, is comprised of both basal and suprabasal cells, which are considered multipotent progenitors capable of renewing the airway epithelium. This schematic depicts how SARS-CoV-2 may bind to ACE2 expressed on the cilia of the nasal cavity following exposure to respiratory droplets or airborne particles.

Article Snippet: For transient transfection of IMCD3 cells, IMCD3 cells were grown to ~80% confluence and transfected with 5 μg of full-length ACE2 plasmid DNA/1 × 10 6 cells using Fugene6 (Promega).

Techniques: