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. 2022 Dec 9;13:7630. doi: 10.1038/s41467-022-34910-5

Fig. 4. Glycosaminoglycans and EGL-modulating enzymes are required for SARS-CoV-2 S-mediated barrier dysfunction.

Fig. 4

A TEER inhibition assay on monolayers of HPMECs or HPMEC/ACE2 treated with heparin (10 µg/mL), S (10 µg/mL), or both simultaneously. TEER readings were taken 24 hpt. Data are from n = 3 biological replicates. B A TEER inhibition assay where monolayers of HPMEC were treated with recombinant hyaluronidase (10 µg/mL), heparin lyases I and III (5 mU/mL each), neuraminidase (1 U/mL), or chondroitinase (25 mU/mL) simultaneously with S (10 µg/mL) treatment. TEER readings were taken 24 hpt. Data are from n = 3 biological replicates. C EGL inhibition assay on HPMEC treated with hyaluronidase (10 µg/mL) or heparin lyases I and III (5 mU/mL each) and simultaneously treated with S (10 µg/mL) and fixed 24 hpt. D Quantification of C from n = 3 biological replicates. Statistics are comparisons of indicated conditions to the S-only control condition. E Representative images from an EGL disruption assay of HPMEC transduced with lentiviruses encoding the indicated gene-targeting guide RNA. Cells were treated with 10 µg/mL S, and sialic acid was visualized by IFA 24 hpt. F Quantification of E from n = 3 biological replicates. Control guide data from this panel are from the same experiment as Fig. S2B. G Same as E and F but using the indicated guide RNAs. Non-target (NT) data are pooled from two cell line replicates. Control guide data from this panel are from the same experiments as Fig. 7J. For all panels, sialic acid is stained with Wheat Germ Agglutinin in green and nuclei are stained with Hoechst in blue with scale bars at 50 µm. MFI is mean fluorescence intensity. Dotted lines are the normalized untreated control conditions. All data are plotted as mean + /− SEM with *p < 0.05, **p < 0.01, ***p < 0.001, and n.s. p > 0.05 by One-Way ANOVA with Tukey’s Multiple comparisons test except for (G) which was analyzed by two-sided unpaired t-test. Statistics in panels A, B, F, and G are comparisons to untreated controls and in panel D are comparisons to the S-only control condition. Source data are provided as a Source Data file.