EMBO/soda-nel huggingface dataset:
The Spectrin cytoskeleton regulates the Hippo signalling pathway. (2015)
Figure 6

Basolateral α-β Spectrins are required for Hippo signalling and membrane tension in the Drosophila follicular epithelium

A Control egg chamber stained for DAPI to mark nuclei.

B Overexpression of Yki3SA stimulates follicle cell proliferation (MARCM clone, GFP positive).

C-F Mutation of βH-spectrin/karst (C) or crb (D) does not increase follicle cell proliferation (GFP negative clone), while mutation of α-spectrin (E) or β-spectrin (F) stimulates follicle cell proliferation (GFP negative clone).

G Control egg chamber stained for Cut, a marker of proliferating cells that is normally down-regulated after stage 6 of oogenesis.

H-K Mutation of βH-spectrin/karst (H) or crb (I) does not increase Cut expression in follicle cells (GFP negative clone), while mutation of α-spectrin (J) or β-spectrin (K) stimulates Cut expression in posterior follicle cells (GFP negative clone).

L Control egg chamber showing ex.lacZ reporter gene expression.

M-P Mutation of βH-spectrin/karst (M) or crb (N) does not increase ex.lacZ expression in follicle cells (GFP negative clone), while mutation of α-spectrin (O) or β-spectrin (P) stimulates ex.lacZ expression in posterior follicle cells (GFP negative clone).

Q Wild-type follicle cells showing normal hexagonal packing of epithelial membranes.

R Mutation of β-spectrin disrupts the normal hexagonal packing, suggesting a role in maintaining membrane tension.

S Clone of β-spectrin mutant cells (GFP negative) showing differential membrane tension at the interface with wild-type cells (GFP positive).

Data information: Scale bars, 25 μm (A–R), 10 μm (S).
The Spectrin cytoskeleton regulates the Hippo signalling pathway. (2015)
Figure 7

Basolateral α-β Spectrins are required to restrict cell proliferation in the Drosophila intestinal epithelium

A Control myo1A.Gal4 UAS.GFP adult midgut stained for phospho-histone H3 to mark mitotic stem cells.

B-E RNAi knock-down of βH-spectrin/karst (B) or crumbs (C) does not increase stem cell proliferation, while RNAi knock-down of β-spectrin (D) or α-spectrin (E) strongly stimulates stem cell proliferation.

F Overexpression of Yki strongly stimulates stem cell proliferation.

G Quantification of the number of PH3-positive mitotic cells. N > 13 samples. Statistical analysis performed using the two-tailed Student's t-test. Error bars show standard deviation. ***P-value ≤ 0.001.

H-K A Hippo reporter DIAP1-HRE-GFP is normally expressed in sporadic stem cells. Its expression is not affected by RNAi knock-down of βH-spectrin/karst (I), but is up-regulated in many stem cells by RNAi knock-down of α-spectrin (J) or by overexpression of Yki (K).

Data information: Scale bars, 100 μm.
The Spectrin cytoskeleton regulates the Hippo signalling pathway. (2015)
Figure 8

Human Spectrin SPTAN1 and SPTBN1 are required for the YAP localisation response to cell density

A Control Caco-2 epithelial cells plated a low density show strong nuclear YAP localisation.

B Control Caco-2 epithelial cells plated at high density show relocalisation of YAP to the cytoplasm.

C, D siRNA knock-down of SPTAN1 (C) or SPTBN1 (D) prevents relocalisation of YAP to the cytoplasm.

E Quantification of YAP localisation in (B–D).

F Western blotting reveals that SPTAN1 or SPTBN1 are required for normal levels of YAP phosphorylation, and of LATS1 phosphorylation.

G siRNAs against SPTAN1 or SPTBN1 efficiently deplete their target proteins.

Data information: Scale bars, 20 μm.
Differential phosphorylation of Clr4Suv39H by Cdk1 accompanies a histone H3 methylation switch that is essential for gametogenesis (2022)
Figure 4

Figure 4 - Restoration of Swi6 binding to pericentromeric repeats rescues the meiotic chromosome segregation phenotype in H3K9me3-depleted cells.

A Comparison of Swi6 localization depending on H3K9 methylation during MI. Exemplary live cell microscopy images of the indicated fission yeast strains are shown. Homothallic strains were spotted on SPAS plates before being mounted on microscopy slides. Swi6, Swi6Chp1-like-CD, and the centromere marker Mis6 were visualized by tagging with yeGFP and mCherry, respectively; Scale bar = 5 µM.

B ChIP-qPCR experiment to assess Swi6 levels at the pericentromeric repeats (cendg) in the indicated fission yeast strains during mitosis. ChIP enrichments were normalized to adh1+ and are shown relative to swi6∆ cells; Error bars, s.d.; n = 4 independent biological replicates. P values were calculated using two-tailed student's t-test.

C Comparison of meiotic chromosome segregation in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary IF microscopy images of the indicated fission yeast strains are shown. Homothallic strains were incubated on SPAS plates. DNA and tubulin were stained with DAPI and anti-TAT-1, respectively. Missegregating chromosome is indicated with a yellow arrow; Scale bar = 5 µM.

D Quantification of meiotic chromosome segregation phenotypes. The data for clr4+ and clr4F449Y is the same as in Fig 3B. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.

E Comparison of co-segregation of centromere I (CenI) during MI in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary live cell microscopy images of the indicated fission yeast strains during MI and MII are shown. Heterothallic strains were crossed and dyads as well as tetrads were subjected to the analysis. Heterozygous CenI-GFP was used to follow the segregation pattern during meiosis. The GFP-signal is additionally displayed in greyscale for better visibility; Scale bar = 5 µM.

F Quantification of CenI-GFP segregation during meiosis. The data for clr4+ and clr4F449Y is the same as in Fig 3D. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.

G Tetrad dissection to asses spore viability in clr4F449Y swi6Chp1-like-CD cells. Exemplary image (left) and quantification of the spore viability (right) are shown. Meiosis was induced in homothallic cells and the resulting spores were dissected. The data for clr4+ and clr4F449Y is the same as in Fig 3F. P values were calculated using Fisher's exact test. Number of dissected tetrads is indicated in each bar.
Differential phosphorylation of Clr4Suv39H by Cdk1 accompanies a histone H3 methylation switch that is essential for gametogenesis (2022)
Figure 6

Figure 6 - Clr4S458 is differentially phosphorylated in a Cdk1-dependent manner in mitosis versus early meiosis.

A Western blot of whole-cell lysates (WCL) using a PhosTag gel for anti-FLAG (top) and a Bis-Tris gel for anti-Act1 detection (bottom), respectively. WCL were prepared from mitotic and meiotic 3xFLAG-clr4+/3xFLAG-clr4+ cultures at the indicated time points.

B Comparing the relative abundance of the differentially phosphorylated Clr4 peptide (DFSPVQSQK) in mitosis and early meiosis by IP-MS; Error bars, s.d.; n = 3 independent biological replicates.

C Western blot of FLAG IPs using a PhosTag gel to asses phosphorylation levels of Clr4 in the indicated homothallic strains.

D Western blot of FLAG IPs in 3xFLAG-clr4+ cdc2-as cells, treated with 2 µM 1-NM-PP1 for the indicated amount of time. The blot was first probed with the phospho-specific anti-pS458 antibody, stripped, and reprobed with an anti-FLAG antibody.

E In vitro kinase assay of recombinant Clr4 and Clr4S58A in presence and absence of human CDK1/Cyclin B. Phosphorylation events were analyzed by mass spectrometry.

F ChIP-qPCR experiments to assess H3K9me2 (left) and H3K9me3 (right)

levels at pericentromeric repeats (cendg) in cdk1+ and cdk1-as cells treated with and without 3 µM 1-NM-PP1. ChIP enrichments were normalized to total H3 levels and adh1+ and are shown relative to untreated cells; Error bars, s.d.; n = 3 independent biological replicates; P values were calculated using two-tailed student's t-test.

Expanded View Figure Legends
RagC Phosphorylation Autoregulates mTOR Complex 1 (2018)
Figure 1

Figure 1. mTORC1 phosphorylates RagC S21

(A) Alignment of the sequence surrounding three phosphorylation sites on RagC with those of known mTORC1 target sites.

(B) The active fragment of mTOR or active Akt2 was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Akt inhibitor GDC-0068.

(C) HEK-293E Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.

(D) HEK-293E cells were deprived of total amino acids for 1hr followed by addition of amino acids for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.

(E) Control or iRapKO MEFs were treated with or without 2 μM 4-Hydroxytamoxifen (4-OHT) for 4 days to induce Raptor knockout. Treated cells were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.

(F) SIN1 WT was expressed in SIN1 KO MEFs, and cells were selected by FACS. Cell lines were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.

(G, H) The active fragment of mTOR was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin. Samples were analysed by Mass Spectrum for pRagC T394 (G) or Western blot for pRagC S21 (H).
RagC Phosphorylation Autoregulates mTOR Complex 1 (2018)
Figure 2

Figure 2. RagC phosphorylation regulates mTORC1 activity

(A) The HA-RagC HeLa Flp-In T-Rex stable cell lines were created (See methods for details) and the expression of RagC WT or mutants was induced by addition of tetracycline overnight. Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3).

(B) Expression of RagC WT, 3A or 3E was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3).

(C) Expression of RagC WT and 3A was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for indicated time. Left: Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Right: Graph shows mean ± S.E.M. of quantitative analyses of the Western blots after normalization for total protein, n=3.

(D) HEK-293E cells stably expressing RagC WT, 3A or 3E were deprived of total amino acids for 1hr followed by addition of amino acids for 10 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, ** p<0.01, two tailed Student's t test, n=3).
RagC Phosphorylation Autoregulates mTOR Complex 1 (2018)
Figure 3

Figure 3. RagC phosphorylation is required for mTORC1 regulation of cell size and autophagy

(A) Expression of RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=4).

(B) Phospho-RagC positively regulates wing compartment size in Drosophila. Over-expression (OE) of dRagC WT, 3A, or 3E in wing posterior compartment using en-Gal4 driver and the posterior compartment sizes were measured in each genotype ((Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. ** p<0.01, *** p<0.001, **** p<0.0001, One-way ANOVA with Bonferroni multiple comparisons, n ≥ 63 flies per condition).

(C) Empty vector (EV), RagC WT, 3A or 3E were transiently expressed in stable RagC knockdown HEK-293E cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, ** p<0.01, **** p<0.0001, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=2 for C2 and n=4 for other conditions).

(D) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Lysates were analyzed by Western blot for phosphorylation of ULK1 and total ULK1.

(E) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. Lysates were analyzed by Western blot for levels of LC3-II and 14-3-3.Graphs show mean ± S.E.M. of quantitative analyses of LC3-II blotting after normalization for 14-3-3. * p<0.05, two tailed Student's t test, n=4.

(F) GFP-LC3 was co-expressed with empty vector, RagC WT, 3A or 3E in HEK-293E cells followed 48 hr later by serum deprivation for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. LC3 and RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm.

(G) Quantitative analysis of GFP puncta (F) (**** p<0.0001, two tailed Student's t test; n ≥ 21). Mean value is shown as a horizontal bar.
RagC Phosphorylation Autoregulates mTOR Complex 1 (2018)
Figure 4

Figure 4. Phosphorylation of RagC alters mTORC1 stability but not the localization of mTOR

(A) Empty vector (EV), HA-RagC WT, 3A or 3E were transiently expressed in HEK-293E cells. RagC and lysosome maker, Lamp1, were monitored by confocal fluorescence microscopy. Scale bar, 10 μm.

(B) Empty vector (EV), HA-RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK-293E cells. mTOR and HA-RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm.

(C) Empty vector (EV), Flag-RagC WT, 3A or 3E were transiently expressed with HA-RagA in HEK-293E cells. Total cell lysates and amounts of mTOR, Raptor and HA-RagA in the Flag-RagC immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (* p<0.05, two tailed Student's t test, n ≥ 3).

(D) Expression of HA-RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells. Total cell lysates (TCL) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (*** p<0.001, Student's t test, n=3).

Expanded View Figure Legends
An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2 (2022)
Figure 1

Figure 1. Aerosol delivery of sACE22.v2.4-IgG1 alleviates lung injury and improves survival of SARS-CoV-2 gamma variant infected K18-hACE2 transgenic mice.

(A) K18-hACE2 transgenic mice were inoculated with SARS-CoV-2 isolate /Japan/TY7-503/2021 (gamma variant) at 1×104 PFU. sACE22.v2.4-IgG1 (7.5 ml at 8.3 mg/ml in PBS) was delivered to the mice by a nebulizer in 25 minutes at 12 h, 48 h, and 84 h post-inoculation. PBS was aerosol delivered as control.

(B-C) Survival (B) and weight loss (C). N = 10 mice for each group. The P-value of survival curve by Gehan-Breslow-Wilcoxon test is shown. Error bars for mouse weight are centered on the mean and show SEM.

(D) Viral load in the lung was measured by RT-qPCR at Day 7. The mRNA expression levels of SARS-CoV-2 Spike, Nsp, and Rdrp are normalized to the house-keeping gene peptidylprolyl isomerase A (Ppia). Data are presented as mean ± SEM, N = 4 mice per group. *, p < 0.05 by unpaired Student's t-test with two sided.

(E) Cytokine expression levels of Tnfa, Ifng, Il1a, and Il1b were measured by RT-qPCR normalized by Ppia. Data are presented as mean ± SEM, N = 4 mice per group. *, p < 0.05 by unpaired Student's t-test with two sided.

(F-G) Representative H&E staining of lung sections at Day 7 post-inoculation for control PBS group (F) and inhalation of sACE22.v2.4-IgG1 group (G). Images at left are low magnifications. Boxed regions (black) are shown at higher magnification on the right. Lungs from 4 independent mice were sectioned, stained, and imaged.
An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2 (2022)
Figure 2

Figure 2. Catalytic activity of sACE22.v2.4-IgG1 contributes to the therapeutic efficacy to mitigate mouse lung injury and improve survival following SARS-CoV-2 gamma infection.

(A) A 7-Methoxycoumarin-4-acetyl (MCA) conjugated peptide is quenched by a 2,4-dinitrophenyl group. ACE2 catalyzed cleavage of the peptide is measured by increased MCA fluorescence. Mutations H374N and H378N generate a catalytically dead sACE22.v2.4-IgG1 protein.

(B-C) Catalytically active sACE22.v2.4-IgG1 (B) and catalytically dead sACE22.v2.4(NN)-IgG1 (C) were immobilized on BLI biosensors that were transferred to solutions of gamma RBD as the soluble analyte (0-60 s) and returned to buffer to measure dissociation (60-360 s). RBD concentrations are indicated on the right of the sensorgrams.

(D-E) Catalytically active sACE22.v2.4-IgG1 and catalytically dead sACE22.v2.4(NN)-IgG1 were aerosolized (7.5 ml protein at 8.3 mg/ml in 25 minutes) and delivered by inhalation to K18-hACE2 transgenic mice at 12 h, 48 h, and 84 h post-inoculation with SARS-CoV-2 gamma variant. 10 mice in each group were observed for survival (D) and weight loss (E). The P-value of survival curve by Gehan-Breslow-Wilcoxon test is shown. Error bars for mouse weight are centered on the mean and show SEM. Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Figure 1.

(F) Viral load in the lung was measured by RT-qPCR at Day 7. The mRNA expression levels of SARS-CoV-2 Spike, Nsp, and Rdrp are normalized to Ppia. Data are presented as mean ± SEM, N = 4 mice per group. ns, no significance by unpaired Student's t-test with two sided.
An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2 (2022)
Figure 3

Figure 3. sACE22.v2.4-IgG1 catalytic activity increases survival of SARS-CoV-2 WA-1/2020 infected K18-hACE2 mice.

(A) K18-hACE2 transgenic mice were inoculated with 1×104 PFU SARS-CoV-2 WA-1/2020 (original variant). Treatment was delayed by 24 hours to allow initial virus replication and evaluate the effects of ACE2 catalytic activity at later stages of disease. sACE22.v2.4-IgG1 was intravenously administered at 15 mg/kg, once per day for 7 days.

(B-C) Survival (B) and weight loss (C) following treatment with an IgG1 isotype control (red) versus catalytically active (blue) and dead (green) sACE22.v2.4-IgG1. N = 8 mice for IgG1 group and catalytic active group, N = 9 for catalytic dead group. Error bars for mouse weight are centered on the mean and show SEM. Gehan-Breslow-Wilcoxon test was applied to analyze the survival curves. P = 0.0099 for catalytic active group compared to catalytic dead group. P = 0.017 for catalytic dead group compared to IgG1 control.

(D-E) Catalytically active sACE22.v2.4-IgG1 (D) and catalytically dead sACE22.v2.4(NN)-IgG1 (E) were immobilized on BLI biosensors and transferred to solutions of RBD (original variant) as the soluble analyte (0-60 s) and returned to buffer to measure dissociation (60-360 s). RBD concentrations are indicated on the right of the BLI sensorgrams.
An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2 (2022)
Figure 4

Figure 4. The engineered decoy tightly binds S of BA.1 omicron and neutralizes infection.

(A) Binding of monomeric sACE2-8his proteins to Expi293F cells expressing S of BA.1 omicron was measured by flow cytometry. Data are mean ± SEM, N = 4 independent experiments.

(B) Binding of dimeric sACE22-IgG1 proteins was compared to the binding of antibodies authorized for therapeutic use in COVID-19 patients. Binding to Expi293F cells expressing BA.1 omicron S was measured by flow cytometry. Data are mean ± SEM, N = 4 independent biological replicates.

(C) Neutralization of BA.1 omicron pseudovirus. sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) were incubated with pseudovirus for 1 h before adding to HeLa-hACE2-11 cells. Infection 48 h later was measured by luciferase reporter gene expression. Data are mean ± SD, N = 3 independent biological replicates.

(D-E) Authentic BA.1 omicron virus (isolate USA/MD-HP20874/2021) was incubated with sACE22-IgG1 (D) or sACE22.v2.4-IgG1 (E) for 1 h and added to Calu-3 cells. Infection 48 h later was measured by RT-qPCR for the viral N gene. 3 µM remdesivir (black columns) is a positive neutralization control. Data are mean ± SD, N = 4 independent replicates.
An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2 (2022)
Figure 5

Figure 5. sACE22.v2.4-IgG1 inhibits SARS-CoV-2 BA.1 omicron replication in vivo.

(A) K18-hACE2 transgenic mice were inoculated intranasally with 1×106 PFU SARS-CoV-2 BA.1 omicron (isolate USA/MD-HP20874/2021) and treated 24 h post-inoculation with IgG1 isotype control or sACE22.v2.4-IgG1 (15 mg/kg/day intravenously, once per day for 7 days).

(B-C) Survival (B) and weight loss (C). N = 8 (IgG1 control) or 9 (sACE22.v2.4-IgG1 treated) mice. Error bars for mouse weight are centered on the mean and show SEM.

(D) Maximal weight loss of mice (compared to Day 0) within 14 days following BA.1 omicron virus infection. Data are presented as mean ± SEM, N = 8 (IgG1 control) or 9 (sACE22.v2.4-IgG1 treated) mice. **, p < 0.01 by two-ways ANOVA, corrected by Tukey.

(E) Viral Spike, Nsp, and Rdrp mRNA levels in lung tissue were measured by RT-qPCR at Days 8 and 14, normalized to Ppia transcript levels. Data are presented as mean ± SEM, N = 4 mice per group. ns, not significant; **, p < 0.01 by two-ways ANOVA, corrected by Tukey.
An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2 (2022)
Figure 6

Figure 6. The engineered decoy tightly binds S of BA.2 and BA.4/BA.5 omicron.

(A) Using flow cytometry, binding to Expi293F cells expressing S of BA.2 omicron was measured for monomeric sACE2-8his proteins. Data are mean ± SEM, N = 3 independent biological replicates.

(B) Binding of antibodies and dimeric sACE22-IgG1 proteins to Expi293F cells expressing BA.2 omicron S measured by flow cytometry. Data are mean ± SEM, N = 4 independent experiments.

(C) BA.2 omicron pseudovirus was incubated with sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) for 1 h and added to HeLa-hACE2-11 cells. Infection was measured after 48 h. Data are mean ± SD, N = 3 independent replicates.

(D-E) Binding of monomeric sACE2-8his proteins (D) and dimeric IgG1 proteins (E) to Expi293F cells expressing BA.4/BA.5 omicron S as measured by flow cytometry. Data are mean ± SEM, N = 3 independent experiments.