Additional file 1 of Advanced pathophysiology mimicking lung models for accelerated drug discovery

Additional file1: Table S1. Summary of primary and secondary antibodies used in IF staining for sectioned models

Single-cell/nucleus analysis of different studies corroborates restricted <i>LRRC15</i> expression in fibroblasts.

(A) UMAP plot of lung single-nucleus RNA seq dataset (Delorey and colleagues). (B) Feature plot and (C) dotplot shows LRRC15 is expressed in Delorey and colleagues fibroblasts. (D) UMAP plot of lung single-nucleus RNA seq dataset (Bharat and colleagues). (E) Feature plot and (F) dotplot shows LRRC15 is expressed in Bharat and colleagues lymphatic endothelial cells and various populations of fibroblasts. The data underlying all panels in this figure can be found in DOI: 10.5281/zenodo.7416876. (TIFF)</p

CRISPR screen analysis and validation.

(A and B) Gene enrichment analysis of screens 2 (A) and 3 (B) performed using MAGeCK. Horizontal dotted line indicates p-value = 0.05. Vertical dotted line indicates log2 fold changes of 2. P-values and LFCs for all genes in screens 2 and 3 are reported in S1 Table. (C-D) Density plot of Z-score (gray) for all sgRNA in (C) screen 2 and (D) screen 3. Blue vertical lines indicate Z-score for ACE2 sgRNAs. Red vertical lines indicate Z-score for LRRC15 sgRNAs. Z-scores calculated as described in methods. (E) Log2 fold changes of all genes in screen 1 vs. log2 fold changes of all genes in screen 2. (F) Log2 fold changes of all genes in screen 1 vs. log2 fold changes of all genes in screen 3. (G) LRRC15 expression of cells in Fig 2E quantified via RT-qPCR. (H) ACE2 expression was not increased in LRRC15 sgRNA transduced cells (quantified via RT-qPCR). (I) The 3 sgRNAs for ACE2 from the Calabrese library used in our screens were transduced into HEK293T-CRISPRa cells, and ACE2 expression was confirmed via qPCR. Only sgRNA3 induced upregulation in ACE2 expression. (J) Transduced cells in (I) were incubated with Spike647 and analyzed via flow cytometry. Only ACE2 sgRNA3 cells showed a significant increase in Spike647 binding. The data underlying all panels in this figure can be found in DOI: 10.5281/zenodo.7416876. (TIFF)</p

Confirmation that LRRC15 binds to SARS-CoV-2 spike protein.

(A) LRRC15 contains 15 leucine-rich repeats, a short cytoplasmic C-terminus, and 2 glycosylation sites. (B) Predicted protein structure of LRRC15 (from alpha fold). (C) LRRC15 is part of the LRR-Tollkin family. (D) Flow cytometry analysis of Alexa Fluor-647 (Spike647) binding in WT HEK293T cells, (E) HEK293T-ACE2, and (F) HEK293T cells with stable expression of ACE2 cDNA and TMPRSS2 cDNA (HEK293T-ACE2-TMPRSS2). Each cell line was transfected with plasmids encoding cDNA for GFP-tagged LRRC15 (transcript 1 or 2) or with empty GFP vector as negative control plasmid. (G). Histogram summary shows MFI of (D–F). (H) Representative images of interaction between LRRC15-GFP and Alexa Fluor 647-conjugated SARS-CoV-2 HexaPro spike protein in HEK293T cells (N = 2). Images were taken at 40× magnification. Green = LRRC15-GFP, red = Spike647, blue = Hoechst-stained nuclei. Scale bar = 25 μm. (I) Immunoprecipitation of LRRC15 with spike protein. Lysates of HEK293T cells transfected with GFP-tagged LRRC15 (transcript 1 or 2, LRRC15_1 and LRRC15_2, respectively) incubated with SARS-CoV-2 HexaPro spike protein were immunoprecipitated using anti-LRRC15 primary antibody. Immunoblots were performed for LRRC15 and for SARS-CoV-2 HexaPro spike. I = input, FT = flow-through, E = elute. The data underlying all panels in this figure can be found in DOI: 10.5281/zenodo.7416876. ACE2, angiotensin-converting enzyme 2; GP5, glycoprotein V platelet; LRRC15, leucine-rich repeat-containing protein 15; MFI, mean fluorescence intensity; SARS‑CoV‑2, Severe Acute Respiratory Syndrome Coronavirus 2; TLR, toll-like receptor; TMPRRS2, Transmembrane serine protease 2.</p

A sensitive FACS-based SARS-CoV-2 spike-binding assay amenable to high-throughput screening.

(A) Schematic of SARS-CoV-2 spike-binding assay. HEK293T cells with stable integration of ACE2 cDNA for overexpression (HEK293T-ACE2) are incubated with Alexa Fluor 488-conjugated SARS-CoV-2 spike protein (Spike488). Spike488-binding cells are then detected by flow cytometry. (B) Representative flow cytometry plots for WT HEK293T and HEK293T-ACE2 incubated with Spike488 (N = 3). See also S1B Fig for gating strategy. (C) Titration of HEK293T-ACE2 (ACE2) cells with WT HEK293T cells. Approximately 1% HEK293T-ACE2 cells showed a difference to baseline non-specific binding. Histogram summary showing MFI of flowed cells. (D) Schematic of CRISPRa system used. (E) Representative plot of flow cytometry for a clonal HEK293T-CRISPRa cell line transduced with NTC sgRNA or ACE2 sgRNA (expression confirmation via RT-qPCR in S1A Fig). The data underlying all panels in this figure can be found in DOI: 10.5281/zenodo.7416876. ACE2, angiotensin-converting enzyme 2; CRISPRa, CRISPR activation; MFI, mean fluorescence intensity; NTC, non-targeting control; SARS‑CoV‑2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA, single-guide RNA.</p

CRISPR activation screen setup.

(A) RT-qPCR of ACE2 expression in SAM clonal cell lines transduced with ACE2 sgRNAs or with HEK293T-ACE2 cells. Results calculated using ΔΔCT method and normalized to NTC sgRNA-transduced HEK293T-CRISPRa cells. (B) FACS gating strategy. Cells were first gated by forward (FSC) and side scatter (SSC) before filtering for singlets. Spike488 fluorescence was gated by comparison with NTC sgRNA transduced cells. Similar strategy was applied to all flow cytometry experiments. (C) FACS results for 3 whole-genome CRISPRa screens with NTC sgRNA-transduced cells as negative controls. For screen 1, cells were incubated with Alexa Fluor 488-conjugated SARS-CoV-2 HexaPro spike (Addgene #154754) and selected on puromycin for 3 days. For screen 2, cells were incubated with Alexa Fluor 488-conjugated SARS-CoV-2 Spike glycoprotein (residues 1–1208, complete ectodomain; gift from Dr. Florian Krammer) and selected on puromycin for 3 days. For screen 3, cells were incubated with Alexa Fluor 488-conjugated SARS-CoV-2 HexaPro spike (Addgene #15474) and selected on puromycin for 8 days. The data underlying all panels in this figure can be found in DOI: 10.5281/zenodo.7416876. (TIFF)</p

LRRC15 is not a SARS-CoV-2 entry receptor but inhibits infection in <i>trans</i>.

(A) IMR90 fibroblasts express LRRC15, quantified via RT-qPCR. N = 3 per cell line. (B and C) TGFβ increased (B) LRRC15 and (C) COL1A1 in fibroblasts, quantified via RT-qPCR. N = 7 for each group. Significance was determined by Mann–Whitney one-tailed test, **p (D) IMR90 fibroblasts expressing LRRC15 bind spike, MFI = mean fluorescence intensity. (E) Fibroblasts do not have innate tropism for SARS-CoV-2 and overexpression of LRRC15 does not mediate infection. Transduction efficiency (luciferase luminescence) was compared to permissive cell line HEK293T-ACE2-TMPRSS2. N = 2 independent replicates for each group. (F) Pooled analysis of 3 independent studies indicate ratio of fibroblasts to epithelial cells in COVID-19 lungs is approx. 2:1 (0.3 in control (n = 19) and 2.06 in COVID-19 (n = 47); unpaired two-tailed t test, p (G) LRRC15 expressing fibroblasts can suppress SARS-CoV-2 spike pseudovirus infection of HEK293T-ACE2-TMPRSS2 cells. Significance was determined by two-way ANOVA, Sidak’s multiple comparison test, **p p N = 6 per condition. (H) LRRC15 expressing fibroblasts can suppress authentic SARS-CoV-2 infection of HEK293T-ACE2-TMPRSS2 cells. Significance was determined by two-way ANOVA, Sidak’s multiple comparison test, *p N = 3 per condition. The data underlying all panels in this figure can be found in DOI: 10.5281/zenodo.7416876. ACE2, angiotensin-converting enzyme 2; COL1A1, collagen type I alpha 1 chain; LRRC15, leucine-rich repeat-containing protein 15; SARS‑CoV‑2, Severe Acute Respiratory Syndrome Coronavirus 2; TMPRRS2, Transmembrane serine protease 2.</p

Ingenuity comparison analysis canonical pathways results.

Canonical pathways output from Ingenuity Comparison Analysis using DRAGEN Differential Expression results for IMR90 TurboGFP vs. IMR90 LRRC15 T1-TurboGFP for input. (XLS)</p

Oligonucleotides for CRISPR activation sgRNA constructs.

Lists oligonucleotides used for generation of CRISPRa sgRNA constructs. Sequences for each sgRNA construct were from either Weismann lab Human Genome-wide CRISPRa-v2 Library (Addgene, #83978) or Calabrese Library Set A (Addgene, #92379). (DOCX)</p

CRISPR activation screen MAGeCK outputs.

Collated output of MAGeCK and MAGeCKFlute pipeline. For each screen, normalized read counts and Z-scores, gene-level summary, sgRNA-level summary, and output of MAGeCKFlute ReadRRA() function are provided. (XLSX)</p