Extended Data Fig. 7: Flow-induced JAG1-NOTCH2 Activation Between VICs and VECs in vitro.
From: APOE–NOTCH axis governs elastogenesis during human cardiac valve remodeling
a. qPCR detection of VEC-related genes in cultured VECs, VICs, and HUVECs. Expression levels are normalized to HUVECs; b. Flow cytometry analysis of PECAM1 (VECs) and COL1A1 (VICs) expression in culture VECs; c. Immunostaining of PECAM1 (VECs) and α-SMA (VICs) on cultured VECs; d. Representative bright field imaging of VECs under static, disturbed, and unidirectional/laminar flow conditions for 48 hours; e. Western blot and statistical analysis of JAG1 and GAPDH in VECs under different flow conditions. Unidirectional shear stress: 8.52 dynes/cm2; f. qPCR analysis of flow responsive genes and JAG1 expression in VECs under static or unidirectional laminar flow; g. Demonstration figure of VECs-VICs co-cultured in no-contacting condition under laminar flow; h. Immunofluorescence staining of α-SMA, NOTCH2, HES1, and EMILIN1 in VICs co-cultured with non-contacting VECs; i. qPCR detection of HEYL and HES1 in VICs co-cultured with VECs under flow conditions in partial-contact manner; j. Demonstration figure of VICs co-cultured with VEC under JAG1 KD in partial-contacting condition under laminar flow; k. qPCR detection of JAG1 in VECs under scramble or JAG1 KD; l-m. Immunofluorescence staining of α-SMA,NOTCH2 (l), and HES1 (m) in VICs co-cultured with partial-contacting VECs with JAG1 KD; n. Demonstration figure of VICs co-cultured with VECs after JAG1 overexpression (OE) in partial-contacting conditions under static flow; o. Western blot and statistical analysis of JAG1 in VECs with empty vector or JAG1 OE; p. Immunofluorescence staining of NOTCH2, NOTCH3, HES1, α-SMA, and PECAM1 in VICs co-cultured with VECs with JAG1 OE under static flow. n = 3 biological repeats. Data shown as ± SEM. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. For a-b: VICs vs. VECs; f, i: Static vs. Laminar flow; P values in a: PECAM1: p < 0.0001, CDH5: p = 0.0002; in b: p < 0.0001; k: scramble vs. JAG1 KD; o: empty vector vs. JAG1 OE. Statistics in a-b, f, i, k, o: Unpaired 2-tailed t-test (two groups), in e: one-way ANOVA followed by Tukey’s test, vs. Static flow. P values in e: Static vs. Disturbed flow: p = 0.6939, Static vs. Unidirectional flow: p = 0.0038; in f: JAG1: p = 0.0331, KLF2: p = 0.0065, KLF4: p = 0.0183, NOS3: p = 0.0129; in i: HEYL: p = 0.0412, HES1: p = 0.0818; in k: p < 0.0001; in o: p = 0.0224. Panels g, j and n created with BioRender.com.
