EMBO/soda-nel huggingface dataset:
Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria (2021)
Figure 6

Figure 6. Partitioning of membrane proteins into fluid domains of phase-separated plasma membranes.

A Time lapse images of E. coli fabA(Ts) cells expressing FOF1 a-mNG. For corresponding controls, see Appendix Fig S10. Cells were grown at 30°C and shifted from 30°C to non-permissive 37°C. See Movie EV2 for full time lapse.

B Images of E. coli fabA(Ts) co-expressing WALP23-mScarlet-I and FOF1 a-mNG grown at permissive 30°C or shifted to non-permissive 40°C. For fluorescence intensity correlations, see Appendix Fig S8D. For corresponding images of E. coli fabA(Ts) cells co-expressing inner membrane marker FOF1 a-mNG and outer membrane marker OmpA-mCherry, see Fig EV5.

C Images of FOF1 a-mCherry-expressing E. coli fabA(Ts) grown at 30°C or shifted to non-permissive 37°C and stained with Laurdan. For fluorescence intensity correlations, see Appendix Fig S8E.

D Growth behaviour of FOF1 a-mNG expressing E. coli fabA(Ts) after shift from 30°C to non-permissive 37°C and upon recovery through exogenous supplementation with UFA oleate (cis-Δ9-C18:1). The corresponding reversible segregation of FOF1 a-mNG is shown above the growth curve. For further controls, see Appendix Fig S11A. For a comparable fluorescence time lapse analysis of mNG-labelled glucose permease PtsG, see Appendix Fig S12.

E Fatty acid composition of E. coli fabA(Ts) cells (same cell batch as in D) upon growth at 30°C, upon depletion of UFA by incubation at 37°C for 120 min and upon recovery by oleate supplementation for additional 120 min. For detailed analyses, see Appendix Fig S11B-D.

Data information: (A-D) The experiments are representative of biological triplicates. (E) The histogram depicts mean and SD of biological triplicates. DIC, differential interference contrast. Scale bar: (A, B, D) 2 µm; (C) 3 µm. Strains used: (A, D, E) E. coli MG4; (B), E. coli MG4/pBH501; (C), E. coli LF6.red.
Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria (2021)
Figure 7

Figure 7. Restricted diffusion of membrane proteins in UFA-depleted E. coli membranes.

A Representative trajectory maps of individual FOF1 a-mNG molecules in E. coli WT and fabA(Ts) cells grown at 30°C and upon shift to non-permissive 37°C for 120 min. See Movie EV3 (WT) and Movie EV4 (fabA(Ts)) for accumulating trajectory maps of FOF1 a-mNG molecules.

B Probability density plots of FOF1 a-mNG jump distances in E. coli WT and fabA(Ts) cells.

C Cumulative probability plots of FOF1 a-mNG jump distances in E. coli WT and fabA(Ts) cells. For an analysis of cell-to-cell heterogeneity of median jump distances of FOF1 a-mNG, see Appendix Fig S13A.

D Apparent lateral diffusion coefficients (Dapp) of FOF1 a-mNG analysed in panel B. An analysis of cell-to-cell heterogeneity of Dapp is shown in Appendix Fig S13B. For a comparable single molecule tracking analysis of transmembrane peptide WALP23-mNG, see Appendix Fig S14 and Appendix Table S3. A detailed analysis of FOF1 a-mNG in an osmotically stabilized fabB15(Ts) mutant (2% KCl) at non-permissive 40°C is shown in Appendix Figs S15, S16 and Appendix Table S4.

Data information: (A) Representative for 3-5 biological replicates. (B, C) Trajectories with ≥5 consecutive frames for each growth condition and strain were pooled from 3-5 biological replicates (n=2345-4468). See Appendix Table S2 for detailed information on cell numbers and jump distances. (D) Median and SD from 3-5 biological replicates, together with P values of a two-sided Wilcoxon rank sum test. Significance was assumed with **** p < 0.0001, *** p < 0.001, n.s., not significant. (A) Scale bar: 1µm. Strains used: (A-D), E. coli MG1, MG4.
Cryo-EM structure of the octameric pore of Clostridium perfringens β-toxin (2022)
Figure 2

Figure 2 Investigating the N-terminal domain of different hemolysins.

A. Characterization of C. perfringens β-toxin (CPB) constructs with different N-termini by gel electrophoretic analysis under denaturing (A) and native (B) conditions. SDS-PAGE gel analyis of CPB samples in cholate (1μg per lane). CPB samples containing cholate were boiled for 5 min at 95°C (+) or not (-).

B. Coomassie stained Blue native PAGE gel (4-16%) of CPB samples (10ug per lane) showing oligomer formation for all different N-termini constructs.

C. Alignment of CPB N-terminus with N-termini of different hemolysins and cryo-EM 2D classification and average of N-terminally tagged WT CPB (1), Δ23CPB (2), δ-toxin N-terminus Δ23CPB (3), C-terminally tagged WT CPB (4), Hla N-terminus Δ23CPB (5) and HlgB N-terminus Δ23CPB (6).

D. Viability of HEK 293FT/CD31-GFP cells (transduced with CD31-GFP) as a percentage of untreated control cells after incubation with indicated concentrations of toxins (24 h, 37°C). Data (technical replicates) are represented as means (n = 4) ± SD.
LincRNA-EPS impairs host antiviral immunity by antagonizing viral RNA-PKR interaction (2022)
Figure 5

Figure 5. Stronger activation of IFNAR downstream signaling independent of IFN-β induction in the lincRNA-EPS-/- macrophages.

A, B RT-qPCR analysis of Ifnb1 mRNA level in the WT and lincRNA-EPS-/- iBMMs infected with VSV (MOI 0.1) for 6 h (A), ELISA analysis of supernatant IFN-β protein after infecting for 8 h (B).

C, D Mx1 (C) and Oas2 (D) mRNA level were measured in the control (EV) and lincRNA-EPS-rescued (lincRNA-EPS) iBMMs after infecting with VSV with different titer for 6 h.

E LincRNA-EPS-stably overexpressed iBMMs (lincRNA-EPS) and corresponding control group (EV) were infected with VSV for different titer (MOI 0.1 and MOI 0.5), Mx1 and Oas2 mRNA level were measured by RT-qPCR after 6 h.

F The basal level of ISGs and proinflammatory genes in the WT and lincRNA-EPS-/- BMDMs (Datasets from the ArrayExpress database under the accession number E-MTAB-4088) were listed by heatmap.

G ChIP assays using anti-H3K4me3 antibody was carried out in WT and lincRNA-EPS-/- iBMMs with or without polyI:C (1 μg/mL) transfection for 4 h, Mx1 and Oas2 enrichment relative to input were measured by qPCR.

H, I WT and lincRNA-EPS-/- iBMMs were infected with VSV (MOI 0.1) for 6 h or 10 h (H) or transfected with polyI:C (1 μg/mL) for 4 h or 8 h (I), phosphorylation and total protein expression were analyzed by Western blot with α-tubulin and β-Actin as loading controls.

Data information: Data of (A-E) and (G) are shown as the mean ± s.d. from three independent experiments, *p < 0.05 and **p < 0.01 by unpaired student t-test. Data of (H, I) are representative results from three independent experiments.
LincRNA-EPS impairs host antiviral immunity by antagonizing viral RNA-PKR interaction (2022)
Figure 6

Figure 6. LincRNA-EPS and viral RNA competitively interact with PKR.

A LincRNA-EPS and negative control RNA (NC) were transcribed in vitro to label desthiobiotin and pulldown proteins from iBMMs lysate, the differential protein bands from silver-stained SDS-PAGE were cut for mass spectrometry analysis. One of the identified proteins from the band in the red box was PKR.

B Western blot for verification of RNA pulldown-MS results with β-Actin as a negative loading control.

C The interaction between PKR and lincRNA-EPS were checked by RIP-qPCR using anti-PKR antibody, and the efficiency of IP was verified by Western blot.

D 20 nM siRNA targeting negative control (NC) and PKR were transfected into iBMMs for 48 h, the protein and mRNA expression level of PKR were tested by RT-qPCR and Western blot, respectively.

E, F VSV titer in the WT and lincRNA-EPS-/- iBMMs transfected with siRNA as described in (D) and infected with VSV (MOI 0.1) for 8 h was measured by plaque assay (E). The alteration rates of viral titers were calculated in lincRNA-EPS-/- iBMMs relative to WT group (F).

G VSV-N and VSV-G mRNA were transcribed from virus particles for RNA-pulldown, the interactions between VSV RNA with PKR protein was checked by Western blot, β-Actin was shown as a negative control.

H LincRNA-EPS was transcribed in vitro and pulled down proteins from iBMMs infected with VSV for 4 h, PKR protein was detected by Western blot and GAPDH was shown as a loading control.

I RNA-pulldown using biotinylated lincRNA-EPS from cell lysates of polyI:C (1 and 2.5 μg/mL) transfected iBMMs for 4 h, PKR protein was detected by Western blot and β-Actin was shown as a loading control.

J HEK293T cells transfected with lincRNA-EPS and Flag-tagged PKR were infected with VSV to detect the PKR-binding lincRNA-EPS and VSV RNA by RIP-qPCR using anti-Flag antibody.

Data information: Data of (C, up panel), (D, left panel), (E, F) and (J) are shown as the mean ± s.d. from three independent experiments, *p < 0.05 and **p < 0.01 by unpaired student t-test. Data of (B), (C, down panel), (D, right panel), (G, H, I) are representative images from three independent experiments.
LincRNA-EPS impairs host antiviral immunity by antagonizing viral RNA-PKR interaction (2022)
Figure 7

Figure 7. LincRNA-EPS inhibits PKR-STAT1-dependent induction of ISGs.

A 20 nM siRNA for negative control (NC) and PKR were transfected into iBMMs following infected with VSV (MOI 0.1) for 4 h or 8 h, phosphorylation and total levels of STAT1, eIF2α and PKR were detected by Western blot and β-Actin was shown as a loading control.

B PKR was transfected into A549 cells for 24 h following stimulated with IFN-β (50 ng/mL) for 1 h to test STAT1 phosphorylation by Western blot and β-Actin was shown as a loading control.

C-H 20 nM siRNA for NC and PKR were transfected into WT and lincRNA-EPS-/- iBMMs following infection with VSV (MOI 0.1) for 4 h, Mx1, Ifit2, and Isg15 mRNA level were measured by RT-qPCR (C, E, and G), and relative basal expression change rates of the antiviral ISGs in lincRNA-EPS-/- iBMMs were calculated compared with WT group (D, F, and H).

I Working model of lincRNA-EPS in resting and viral infected WT or lincRNA-EPS-/- cells.

Data information: Data of (A, B) are representative images from three independent experiments. Data of (C-H) are shown as the mean ± s.d. from three independent experiments, *p < 0.05 and **p < 0.01 by unpaired student t-test.

EXPANDED VIEW FIGURE LIGENDS
Xanthomonas campestris attenuates virulence by sensing light through a bacteriophytochrome photoreceptor (2016)
Figure 3

Figure 3. X. campestris extracellular endoglucanase production is regulated by XccBphP.

(A) Five µL supernatants from wild-type, XccbphP, pXccBphP and pC13 strains bacterial cultures (OD600 = 1) grown under light or dark conditions were plated onto PYM-carboxymethyl cellulose (CMC) agar plates and revealed with Congo red staining (n = 2 replicates). The extracellular β-1,4-endoglucanase production levels correlated with CMC degradation halo radiuses (top panel). Halo measurements are presented in the bottom panel. Data derive from 10 independent experiments. (B) The extracellular β-1,4-endoglucanase activity from bacterial cultures supernatants (OD600 = 1) was determined by a colorimetric assay in solution (n = 2 replicates). Wild-type, XccbphP, pXccBphP and pC13 strains were assayed under normal laboratory conditions. Data derive from 3 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).
Cyclin D1 controls development of cerebellar granule cell progenitors through phosphorylation and stabilization of ATOH1 (2021)
Figure 2

Figure 2 Requirement of CCND1 and CDK4 in the AT-ND transition

A. Normalized expression levels of Ccnd1 in each cluster (Fig 1C) as indicated by violin plots.

B. Immunostaining of P5 cerebellum with indicated antibodies. Boxplots of the intensity of CCND1 immuno-signal (B'''', normalized to the mean intensity of AT+GCPs). We measured the intensities of CCND1 in ATOH1+/NEUROD1- cells and NEUROD1+/ ATOH1- cells. ATOH1+/ NEUROD1+ cells were not included in this quantification. These boxplots are showing the median (central band), 25% / 75% quartiles (boxes) and the ranges (lower whiskers represent the regions of lower box - 1.5 * IQR (inter-quartile range), upper whisker represent the regions of upper box - 1.5 * IQR). Wilcoxon test were performed between AT+GCPs and ND+GCPs. ***p < 0.001.

C-F. Ccnd1 KD analysis. (C, D) Ccnd1 shRNAs together with H2B-EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (Cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (C) and Ccnd1 KD (D). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (E) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were decreased in the Ccnd1 KD mice. (F) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=3) and Ccnd1 KD (sh#1: N=3, sh#2: N=3) mice. Bars and error bars are representing as Mean ± SEM. ***p < 0.001, N.S.: Not Significant. Number of cells: 52, 64 and 107 cells from N=3 mice (scramble), 72,79 and 75 cells from N=3 mice (sh#1), 84, 70 and 40 cells from N=3 mice (sh#2).

G-K. Over-expression analysis of Ccnd1 with or without Cdk4. (G, H, I) Ccnd1 and Cdk4 expression plasmids were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (G), CCND1 over expression (H) and CCND1 and CDK4 over expression (I). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (J) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were not significantly changed between Control and CCND1. Forced expression of CCND1 and CDK4 (4D) significantly increased the proporations of AT+GCPs compared to Control. (K) Proportions of KI67+ cells (total GCPs) were not significantly changed. Tukey-Kramer test was performed on Control (N=6), CCND1 (N=4), CCND1 and CDK4 (N=3). Bars and error bars are representing as Mean ± SEM. **p < 0.01, N.S.: Not Significant. Number of cells: 52, 64, 107,244,127 and 146 from N=6 mice (Control), 126, 137, 80 and 336 from N=4 mice (Ccnd1), 212, 143 and 38 from N=3 mice (4D).

Data information: Scale bars: 20µm (B, G-I), 50µm(C, D)
Cyclin D1 controls development of cerebellar granule cell progenitors through phosphorylation and stabilization of ATOH1 (2021)
Figure 3

Figure 3 CCND1/CDK4 phosphorylates and stabilizes ATOH1 protein

A, B. Cycloheximide (CHX) assay. (A) N2a cells transfected with Atoh1-GST, Ccnd1 and Cdk4 plasmids 2 days prior were harvested after 0, 4 and 8 hr treatments of 50ug/mL CHX and lysates were subjected to immunoblotting with anti-Atoh1 or β-actin as a housekeeping control. (B) Quantification of relative levels of ATOH1 to the β-ACTIN. N2a transfected with only Atoh1-GST is referred as WT and N2a transfected with Atoh1-GST, Ccnd1 and Cdk4 plasmids is referred as 4D. Same experiments were performed three times. Error bars are representing as Mean ± SEM.

C. N2a cells transfected with Atoh1-GST plasmids were treated with DMSO(Control), Palbociclib (Palb) or Palb and 10 uM MG132 (MG132) for 6hrs before harvest. Lysates of N2a were subjected to immunoblotting with anti-Atoh1 or β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between Control, Palb, MG132. Bars and error bars are representing as Mean ± SEM from three trials of each condition. ***p < 0.001, *p < 0.05, N.S.: Not Significant. Three slots of each experimental condition represent the three biological replicants of each condition.

D. in vitro phosphorylation assay of ATOH1. GST-tagged full length ATOH1 (FL), N-terminus ATOH1 including the bHLH domain (N) and C-terminus of ATOH1 downstream of the bHLH domain (C) were purified from N2a cells using glutathione-beads. ATOH1 proteins were incubated with either Casaine kinase 1α (CK1α) or CCND1 and CDK4 in the presence of [γ-32P]-ATP. Phosphorylation signals detected with FL, N and C by the phosphorylation of CK1α (lanes 1, 2, 3) are indicated by white arrowheads. Phosphorylation signals were also detected with FL and C by the phosphorylation of CCND1-CDK4 (lanes 5, 6, 7). Phosphorylation signals of N with CCND1-CDK4 were not detected. Strong CK1α self-phosphorylation signals were observed (lane 4).

E. Phosphorylation sites of ATOH1-GST purified from N2a cells were determined by LC-MS/MS. 5 phosphorylation sites (Y78, S82, S309, S311, S328) were identified.

F. In vitro kinase assay with either wild type ATOH1 (WT) or non-phosphorylated form of the Serine-to-Alanine 309 (S309A). WT or S309A were incubated with CCND1 and CDK4 (4D) in the presence of [γ-32P]-ATP. While WT with CCND1 and CDK4 gave rise to strong signals (lanes 1,2 white arrowhead), S309A showed no obvious signal (dashed arrowhead indicated the predicted size of ATOH1).

G. Atoh1 expression plasmids were transfected into N2a cells with Ccnd1 and Cdk4 expression plasmids (4D). N2a cells were treated with 10 nM, 100 nM, 1000 nM Palb for 6 hrs before harvest. Lysates from N2a cells were subjected to immunoblotting with anti-Atoh1, p-S309-Ab and β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between 0, 10, 100, 1000nM. Bars and error bars are representing as Mean ± SEM from three trials of each condition. **p < 0.01, *p < 0.05, N.S.: Not Significant.
Cyclin D1 controls development of cerebellar granule cell progenitors through phosphorylation and stabilization of ATOH1 (2021)
Figure 6

Figure 6 Dynamic expression of ATOH1, CCND1 and PROX1

A. P5 or P10 mouse cerebella were electroporated with H2B-GFP plasmids and immunostained with anti-Atoh1 (magenta) the following day (P6 or P11). Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. The proportions of ATOH1-positive cells(AT+GCPs, A'). Bars and error bars are representing as Mean ± SEM. Student T-test was performed between P6 mice electroporated at P5 (P5-6) and P11 mice electroporated at P10 (P10-11). ***p < 0.001. Number of cells: 108, 72 and 104 cells from N=3 mice (P5-6), 182, 45 and 61 cells from N=3 mice (P10-11).

B-F. AT-ND transition in cultured GCPs. (B-E) GCPs were extracted from P5 (P5 GCPs) or P10 (P10 GCPs) mouse cerebella and immunostained with anti-Neurod1 (green) and anti-Ki67 (magenta) at DIV1 and DIV2. (F) Proportions of NEUROD1- cells (AT+GCPs) among total KI67-positive cells. Bars and error bars are representing as Mean ± SEM. Tukey-Kramer test was performed. **p <0.01, N.S.: Not Significant. Number of cells: 95,194 and 53 cells from N=3 mice (P5DIV1), 543, 238 and 550 cells from N=3 mice (P5DIV2), 121, 242 and 232 cells from N=3 mice (P10DIV1), 110,137 and 210 cells from N=3 mice (P10DIV2).

G. Normalized expression levels of Ccnd1 (G), Prox1 (G') and Gapdh (G'') transcripts were compared between Atoh1-positive cells extracted from the scRNA-seq data of P5 and P14 cerebellum. Wilcoxon test between n=1602 cells (P5 Atoh1-positive cells) and n=274 cells (P14 Atoh1-positive cells). ***p <0.001, N.S.: Not Significant.

H-J. Whole cerebellum lysates of P6, P9 and P12 mice were subjected to immunoblotting with anti-pAtoh1 S309, Atoh1, Ccnd1, Prox1, β-actin. (I) The protein levels of CCND1 and PROX1 normalized by β-ACTIN. (J) Quantification of p-ATOH1 S309 normalized to the total level of ATOH1.

K. ChIP assay for H3K9 histone-acetylation of Ccnd1 promoter in cultured GCPs extracted from P6 or P9 mouse cerebellum. Student T test between P6 GCPs (N=4) and P9 GCPs (N=4). *p <0.05. Bars and error bars are representing as Mean ± SEM.

L-P. Over-expression analysis of WT or S309D form of Atoh1. (L) Expression plasmids of either WT or S309D form of Atoh1 were introduced to P10 mice EGL and immunostaining for KI67 (magenta) was performed in P16 mouse cerebellum. Nuclei of electroporated cells were detected by GFP (green). Images of H2B-GFP (M, Control), WT(N) and S309D (O). Arrows indicate the cells of interest where GFP signals are co-localized with KI67. (GCPs). (P) Proportions of KI67+ cells among the electroporated cells. Tukey-Kramer test among Control (N=3), Atoh1 WT (N=3) and Atoh1 S309D (N=3). Bars and error bars are representing as Mean ± SEM. *p <0.05. Number of cells: 43, 68 and 46 from N=3 mice (Control), 104, 215 and 157 cells from N=3 mice (WT), 242, 119 and 111 cells from N=3 mice (S309D).

Data information: Scale bars: 50µm (A), 20µm (B-E, M-O)
LY6E impairs coronavirus fusion and confers immune control of viral disease (2020)
Figure 1.

ISG screen identifies LY6E as a potent coronavirus restriction factor.a, Duplicate screens depicting HCoV-229E infection (24 hours post-infection) in Huh7 cells ectopically expressing ISGs. b, HCoV-229E infection of Huh7 cells expressing LY6E or control vector (Empty). Blue: DAPI, green: HCoV-229E N protein, red: TagRFP encoded in SCRPSY vector. c-h, Effect of LY6E expression on diverse coronaviruses. Stably transduced cells were infected with HCoV-229E (c), HCoV-OC43 (d), MERS-CoV (e), SARS-CoV (f), SARS-CoV-2 (g), MHV-Gluc (h). i, Western blot of WT A549 or two LY6E KO clones (#3, #4). j, HCoV-229E infection of WT or LY6E KO A549. k, Western blot of WT or LY6E KO A549 reconstituted with CRISPR-resistant LY6E (CR LY6E) or control vector (Empty). l, HCoV-229E infection of CR LY6E-reconstituted WT or LY6E KO A549. Viral replication readouts included: titer determination by plaque assay (c), infectivity by nucleoprotein staining and quantitation by flow cytometry (d), limiting dilution assay (e,f,g), luciferase activity shown as relative light units (RLU) (h,j,l). Data represent average of independent biological replicates, n=3 (c-d,f-h), n=4 (e,j,l). In b, scale bars are 200 µM. Statistical significance was determined by unpaired student's t-test with Welch's correction (c-d, f-g), one-tailed Mann-Whitney U test (e), ratio paired student's t-test (g), 2way ANOVA followed by Sidak's or Dunnett's multiple comparison test (j,l). Error bars: SD. P values: c, ** p=0.0088; d, *** p=0.0001; e, * p=0.0143; f, * p=0.0286; g, * p=0.0101; h, *** p=0.0010; j, ** p=0.0073, p=0.0033; l, ns=0.0740, ** p=0.0013, *** p=0.0001.
LY6E impairs coronavirus fusion and confers immune control of viral disease (2020)
Figure 2.

LY6E inhibits viral membrane fusion and syncytia formation.a-d, VSV pseudoparticles (PP) harboring spike proteins from HCoV-229E (a), MERS-CoV (b), SARS-CoV (c), SARS-CoV-2 (d) were inoculated on LY6E- or empty vector-expressing cells. Virus entry efficiency was quantified by measuring virus-encoded luciferase reporter activity. e, VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP (VSV*ΔG(CoV S)) were inoculated with LY6E- or empty vector-expressing Huh7. Syncytia formation was analyzed by immunofluorescence microscopy. Blue: DAPI, green: GFP, red: TagRFP inserted in SCRPSY vector. f, Quantification of VSV*ΔG(CoV S) induced syncytia depicted as percentage syncytia area. Three independent areas were analyzed per biological replicate (circle, square, triangle). g, Schematic depiction of the cell-cell fusion assay created with BioRender. h, Cell-cell fusion was measured by co-incubating cells transfected with plasmids encoding CoV S proteins and a split-luciferase construct (DSP1-7) together with CoV permissive LY6E-expressing or control cells transfected with the second fragment of split-luciferase (DSP8-11). Data represent averages of independent biological replicates, n=8 (a,b), n=6 (c,d), n=3 (f), n=4 (h). Statistical significance was determined by unpaired student's t-test with Welch's correction (a-d) and 2way ANOVA followed by Sidak's multiple comparison test (f,h). Outliers were removed using Grubbs outlier test (a). In e, scale bar is 20 µM. Error bars: SD. P values: a, **** p=<0.0001; b, **** p=<0.0001; c, **p=0.0066; d, **** p=<0.0001; f, **** p=<0.0001; h, ns p=0.9975, ** p=0.0054, ns p=0.1520, 0.9892.