Fig 1: Schematic of study design.Plasma and serum samples were obtained from multiple patient cohorts across two UK-based university hospitals, including 123 COVID-19 patients: 78 SARS-CoV-2 positive patients in ICU were sampled at multiple time points over a 2-week period and compared to hospitalized non-ICU SARS-CoV-2 positive patients (n = 45). We used non-COVID-19 ICU patients (n = 25) and patients before undergoing elective cardiac surgery (n = 30) as controls. Patient samples were assessed for SARS-CoV-2 RNAemia, antibody responses, and protein changes in the circulation by data-independent acquisition (DIA) mass spectrometry (MS) analysis. Plasma protein interactions with SARS-CoV-2 spike glycoprotein were determined using a pulldown assay followed by data-dependent acquisition (DDA) MS analysis. Functional effects of LGALS3BP were assessed in two assays: SARS-CoV-2 spike-mediated cell-cell fusion (syncytia formation) and cell entry through SARS-CoV-2 spike pseudoparticle assays.
Fig 2: Extrinsic effect of LGALS3BP on ventral progenitor fate and neuronal specification.(A and B) Experimental setup for media exchange in vCOs (A) and for the generation of vMCOs (B). (C to G) Micrograph of D60 CTRL (C), E370K (D), E370K-vCOs fed with CTRL CM (E), and vMCOs (F) and (G) immunostained for EOMES. (H) Quantification of the percentage of EOMES+ cells in vCOs and vMCOs normalized by DAPI in the analyzed GE unit. (I to M) Micrograph of D60 CTRL (I), E370K (J), E370K-vCOs fed with CTRL CM (K), and vMCOs (L) and (M) immunostained for TBR1. (N) Quantification of the percentage of TBR1+ cells in vCOs and vMCOs normalized by DAPI in the analyzed GE unit. (O to S) Micrograph of D60 CTRL (O), E370K (P), E370K-vCOs fed with CTRL CM (Q), and vMCOs (R) and (S) immunostained for SATB2. (T) Quantification of the percentage of SATB2+ cells in vCOs and vMCOs normalized by DAPI in the analyzed GE unit. (U to Y) Micrograph of D60 CTRL (U), E370K (Y), E370K-vCOs fed with CTRL CM (W), and vMCOs (X) and (Y) immunostained for GAD67. (Z) Quantification of the percentage of GAD67+ signal in vCOs and vMCOs normalized by area [for (U) to (W)] and by GFP+ area [for (X) and (Y)] in the analyzed GE unit. Each individual data point represents one GE unit (ventricle). Data are shown as means ± SEM Significance was based on the Kruskal-Wallis multiple comparison test; *P = 0.029 and ****P < 0.001. Scale bars, 50 µm.
Fig 3: Extrinsic function of LGALS3BP in progenitor specification and neuronal migration.(A) Schematic of isolation of EVs from vCOs. (B) LGALS3BP immunoelectron micrographs in EVs collected from COs. (C) Schematic of 20D E370K-vCO dissociation and EV treatment. (D and E) Micrograph of E370K cells treated with E370K-vEVs (D) and CTRL-vEVs (E) immunostained for TBR1. (F) Quantification of TBR1+ cells in (D) and (E) normalized by DAPI. (G and H) Micrograph of E370K cells treated with E370K-vEVs (G) and CTRL-vEVs (H) immunostained for SATB2. (I) Quantification of SATB2+ cells in (G) and (H) normalized by DAPI. (J and K) Micrograph of ventral E370K cells treated with E370K-vEVs (J) and CTRL-vEVs (K) immunostained for GAD67. (L) Quantification of GAD67+ signal in (J) and (K) normalized by area. (M) Schematic of 20D CTRL-vCO dissociation and EV treatment. (N and O) Micrograph of ventral CTRL cells treated with CTRL-vEVs (N) and E370K-vEVs (O) immunostained for GAD67. (P) Quantification of GAD67+ signal in (N) and (O) normalized by area. (Q) Venn diagram of proteins detected in CTRL-vEVs and E370K-EVs. (R) Volcano plot of DE proteins in E370K-vEVs, plotting the negative log10 q values (FDR) of all proteins against their log2 fold change. (S) Schematic of treatment of NPCs with CTRL-vEVs and E370K-vEVs. (T to W) GO terms of up-regulated pathways (T), and gene networks of WNT (U), NOTCH (V), and axon guidance (W) pathways in NPCs after E370K-vEVs exposure. Every dot in the plots refers to an analyzed field of view. Significance was based on the unpaired t test; *P < 0.05, **P < 0.01, and ****P < 0.001. Scale bars, 100 nm (B) and 50 µm (D), (E), (G), (H), (J), (K), (N), and (O).
Fig 4: LGALS3BP overexpression impairs SARS-CoV-2 spike-mediated syncytia formation and cellular uptake of SARS-CoV-2 spike-pseudoparticles.a Schematic representation of the SARS-CoV-2 spike-mediated cell-cell fusion assay. b–d Vero and HEK293-ACE2 cells were transfected either with pcDNA3 (plasmid backbone), pmCherry (plasmid coding mCherry), pLGALS3BP (plasmid coding LGALS3BP), siACE2 (siRNA targeting ACE2), or siNT1 (non-targeting siRNA), followed by transfection of pAAV-Spike (plasmid coding SARS-CoV-2 spike) 24 h later. After 20 h, cells were stained with anti-LGALS3BP (violet), anti-Spike (green), and DAPI for nuclei (blue). Representative images are shown in (b), and quantifications are shown in (c) for Vero cells and in (d) for HEK293-ACE2 cells. Data (mean ± standard deviation; n = 6; Mann–Whitney U test) are plotted as the percentage of fused cells (syncytia) normalized to the total number of cells. Scale bars in (b) represent 100 µm. e Schematic representation of the SARS-CoV-2 spike/VSV-G pseudoparticle transduction assay. f–h HEK293-ACE2 cells were transfected either with pcDNA3, pLGALS3BP, siACE2, or siNT1, followed by the addition of spike- or VSV-G pseudoparticles carrying a GFP reporter 24 h later. After 36 h, cells were stained with anti-LGALS3BP (red), anti-GFP (green), and DAPI for nuclei (blue). Representative images are shown in (f) and quantifications are shown in (g) for spike-pseudoparticles (mean ± standard deviation; n = 6; Mann–Whitney U test) and in (h) for VSV-G pseudoparticles (mean ± standard deviation; n = 3). Data are plotted as the percentage of GFP-positive cells normalized to the total number of cells. Scale bars in (f) represent 200 µm. All statistical analyses are two-tailed.
Fig 5: Extrinsic effect of LGALS3BP on ventral progenitor fate and neuronal specification.(A) Schematic of the experimental setup for the generation and processing of vMCOs. After 60 days of culture, vMCOs are dissociated into single cells, and CTRL- and GFP-labeled E370K cells are sorted through fluorescence-activated cell sorting (FACS) and processed for scRNA-seq. The image was partially generated using BioRender. (B) UMAP visualization of scRNA-seq data of D60 vMCOs (n = 4898 cells from a pool of five organoids). (C) UMAP visualization of scRNA-seq clusters of D60 vMCOs. Each color cluster represents a different cell population. (D) Feature plot depicting the expression of progenitor markers (TOP2A and PAX6), IPs markers (NKX2-1, ASCL1, and GLI3), and neuron markers (DLX5, MAP2, TBR1, NEUROG1, and SLC17A6) in D60 vMCOs. (E) Volcano plot showing the genes rescued in progenitors in vMCOs, plotting the negative log10 P.adj of all genes against their log2 fold change (vMCOs-E370K versus vMCOs-CTRL). Rescued genes are labeled in gray, significantly up-regulated in red, and down-regulated in blue (P.adj < 0.01). (F) Volcano plot showing the genes rescued in IP and MGE IP in vMCOs, plotting the negative log10 P.adj of all genes against their log2 fold change (vMCOs-E370K versus vMCOs-CTRL). Rescued genes are labeled in gray, significantly up-regulated in red, and down-regulated in blue (P.adj < 0.01). (G) Volcano plot showing the genes rescued in neurons in vMCOs, plotting the negative log10 P.adj of all genes against their log2 fold change (vMCOs-E370K versus vMCOs-CTRL). Rescued genes are labeled in gray, significantly up-regulated in red, and down-regulated in blue (P.adj < 0.01).
Supplier Page from Abcam for Anti-LGALS3BP antibody [EPR21757-33]