Additional file 1 of Maternal fiber-rich diet promotes early-life intestinal development in offspring through milk-derived extracellular vesicles carrying miR-146a-5p
Creators: name: Lu, Dongdong
name: Liu, Yisi
name: Kang, Luyuan
name: Zhang, Xiangyu
name: Hu, Jie
name: Ye, Hao
name: Huang, Bingxu
name: Wu, Yujun
name: Zhao, Jinbiao
name: Dai, Zhaolai
name: Wang, Junjun
name: Han, Dandan
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Chemical Sciences not elsewhere classified
subject: Science Policy
subject: Developmental Biology
subject: Hematology

description: Supplementary Material 1: Table S1. Ingredients and nutrient compositions of experimental diets. Table S2. The sequences of primer for the RT-qPCR measurement of miRNAs and mRNAs. Table S3. The antibodies, reagents, commercial assays, software, and other resource used in this study. Table S4. The effects of maternal dietary RS intervention on growth performance of suckling piglets. Table S5. The effects of maternal dietary Resistant starch intervention during lactation on milk quality of sows. Figure S1. Characterizations of mEVs and mEVs-removed supernatant isolated from sow milk. (A) Physical characteristics observed by TEM, scale bars = 200 nm. (B) The size distribution of mEVs is measured by Nanosight. (C) The marker protein expression in mEVs and mEVs-removed supernatant. (D) The concentration of particles of mEVs and mEVs-removed supernatant. Figure S2. Gradient experimental design to explore the optimal treatment concentration and treatment time of mEVs in IPEC-J2 cell line. (A) The gradient experimental design for optimal treatment time. The spherical test results were not satisfied (p = 0.01), the difference between groups was significant (p = 0.03), and interaction did not exist (time*group = 0.15). The OD of cell culture at each time point was analyzed separately and data are expressed as means ± SD. (B) The gradient experimental design for optimal concentration, data are expressed as means ± SEM. Differences in superscript letters for the peer data indicate that the difference is significant (p &lt; 0.05). Figure S3. The miRNA expression profiles in two kinds of mEVs and their effects on the proliferation of IPEC-J2 cells. (A) The volcanic map of expression of miRNAs between the CmEVs and RmEVs. Red, upregulated in RmEVs. Blue, upregulated in CmEVs. (B) KEGG pathway analysis of target genes of miRNAs differently expressed in CmEVs and RmEVs. (C-I) The relative expression of miR-6516, miR-223, miR-1285, miR-9841-3p, miR-18a, miR-146a-5p, miR-142-5p in CmEVs, RmEVs, and their supernatants measured by RT-qPCR. (J, K) The cell proliferation of IPEC-J2 after transfected by mimics, inhibitors, and NC of miR-6516, miR-223, miR-1285, miR-9841-3p, miR-18a, miR-146a-5p, and miR-142-5p, respectively, then measured by MTT assay. Data are expressed as means ± SEM. *p &lt; 0.05, **p &lt; 0.01. Figure S4. The high-resolution images of Western blot obtained by the WESTM System. (A) IPEC-J2 cells transfected with miR-146a-5p mimics. (B) IPEC-J2 cells were transfected with pcDNA3.1-NEDD4L or pcDNA3.1-control, then treated with MG132 or DMSO. (C) IPEC-J2 cells were transfected with miR-146a-5p mimics or NC, then treated with MG132 or DMSO. (D) HEK293 cells were transfected with constructs expressing Flag-NEDD4L, HA-Ubiquitin (HA-UB), MYC-DVL2, and miR-146-5p mimics or mimics-NC, followed by anti-MYC IP detection. Figure S5. Overexpression of miR-146a-5p increases the proliferation and renewal of intestinal cells in mice model (n = 8). (A) Representative images of jejunum stained for HE, EdU, β-catenin, CCND1, and cleaved-caspase 3. Scale bars: 50 µm. (B, C) The villi length and crypt depth of jejunum. (D-G) The ratio of positive cells in jejunum for EdU, β-catenin, CCND1, and cleaved-caspase3. Data are expressed as means ± SEM. *p &lt; 0.05, **p &lt; 0.01