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. 2022 Feb 17;28(2):283–294. doi: 10.1038/s41591-022-01682-w

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a, Diagram of the experiment carried out to examine antibody-mediated calcification of BHV in naïve mice. BHV tissue discs (6 mm) were preincubated with affinity-purified human anti-Neu5Gc IgG or PBS carrier control, then implanted into Neu5Gc-deficient mice (Cmah^−/−); the calcium in discs was measured after one month by atomic absorption spectrometry. b, Calcium deposition quantified in explanted discs (mean ± s.e.m.; two-tailed nonparametric Mann–Whitney U-test; P = 0.0134; n = 5 BHVs; 2 groups (control/treatment), n = 8 mice per group; implanted 4 BHV discs per mouse) c, Diagram of the experiment carried out to examine antibody-mediated calcification of BHVs in immunized Cmah^−/− mice. Mice were immunized with Neu5Gc⁻/Neu5Gc⁺ glyconanoparticles (purple/light blue); then, the immunized-serum pretreated BHV discs were implanted and calcium was quantified after one month. d, Anti-Neu5Gc IgG (light blue) or control IgG (purple) responses in immunized mice were confirmed by glycan microarray analysis at the indicated time points (immunization; left panel; two-way ANOVA with Bonferroni multiple comparisons test, P < 0.0001); then, tissues discs were implanted in preimmunized mice and calcium levels were measured in explanted discs (calcification; right panel; n = 3 tissues of BHVs or porcine αGal/Neu5Gc-deficient (Ggta1^−/−/Cmah^−/−; porcine double knockout); n = 8 mice; 5 discs per mouse; mean ± s.e.m.; unpaired two-tailed t-test; BHV-1, St Jude Medical Epic porcine aortic valve, *P = 0.0175; BHV-2, Carpentier–Edwards bovine pericardium, **P = 0.0032). Glutaraldehyde-treated porcine double knockout pericardium was similarly implanted in preimmunized mice and calcification measured (mean ± s.e.m.; immunization: two-way ANOVA with Bonferroni multiple comparisons test, P < 0.0001; calcification: unpaired two-tailed t-test, NS, P = 0.0998). e, Neu5Gc-containing porcine pericardium (Ggta1^−/−; Gal knockout; glutaraldehyde-treated) was implanted into control Neu5Gc⁻-immunized mice (n = 2; S-0) or into Neu5Gc⁺-immunized mice that had low or high anti-Neu5Gc IgG response according to glycan microarray analysis (n = 3; low: S-1, S-2; high: S-3); then, calcification was measured after 1 month (4 discs per mouse; thick broken line, median; mixed-effects analysis with Dunnett multiple comparisons test; immunization: *P = 0.0107, ***P = 0.0008; calcification: *P = 0.0183).