Extended Data Fig. 4: Overview of Elastin-related Staining and scRNA-seq for PV with Pulmonary Stenosis.

a. Left: Verhoeff staining of pulmonary valves from the other two healthy control and age-matched Pulmonary Stenosis (PS) patients. The black staining within the dashed area indicated the elastin fiber. Right: Quantification of positive staining area of elastin fiber within elastin layer. Age of the sample were from 5-months to 10-years; b. Immunofluorescence staining of ELN, APOE, EMILIN1, and LOXL1 in pulmonary valves from control and PS. White dashed boxes corresponded to the zoom-in figures shown in Fig. 3b. n = 3 different valves; c. UMAP projection of valve cell types within pulmonary valves (PVs) from one healthy control and one patient with PS; d. Feature plots of representative marker genes within each cell type and subtype from c; e. Feature plots of representative marker genes of VIC subtypes comparing PVs from one control and one PS; f. Heatmap demonstrating the gene expression changes of elastase (MMPs) and elastase inhibitors (TIMPs) within each PV-VIC subcluster; g. qPCR of MMPs and TIMPs gene expressions in cultured human VICs comparing scramble vs. APOE KD. n = 3 biological repeats. Data shown as mean ± SEM. ns p > 0.05, *p < 0.05. For a: control vs. PS; For g: scramble vs. APOE KD. Statistics in a, g: Unpaired 2-tailed t-test (2 groups). P value in a: p = 0.0407; in g: MMP1: p = 0.2031, MMP2: p = 0.0649, MMP3: p = 0.1083, MMP9: p = 0.0635; MMP12: p = 0.1017, MMP13: p = 0.2405, MMP14: p = 0.2096, TIMP2: p = 0.0198, TIMP3: p = 0.0313, TIMP4: p = 0.8384.