PepT Expression Helps Maintain Intestinal Homeostasis by Mediating the Differential Expression of mirnas along the Crypt-Villus Axis Yuchen Zhang,*, Emilie Viennois, Mingzhen Zhang, Bo Xiao, 3, Moon Kwon Han, Lewins Walter, Pallavi Garg 4, Didier Merlin, Center for Diagnostics and Therapeutics, Institute for Biomedical Sciences, Georgia State University, Atlanta, Georgia, 33, USA Atlanta Veterans Affairs Medical Center, Decatur, Georgia, 333, USA 3 Institute for Clean Energy and Advanced Materials, Faculty for Materials and Energy, Southwest University, Chongqing, 475, P. R. China 4 Department of Biology, Georgia State University, Atlanta, Georgia, 33, USA * Corresponding author: Yuchen Zhang, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, Tel.: + (44) 43 559; Fax: + (44) 43 358; Email: yzhang4@gsu.edu
Supplementary Figure. Expression of PepT and Lgr5 in isolated epithelial cells of the jejunum from BL-6 WT mice. Total RNAs were extracted from the different fractions collected from BL-6 WT mice using the low-temperature method, and the expression levels of PepT (as a villus marker) and Lgr5 (as a crypt marker) were assessed by qrt-pcr. Supplementary Figure. Expression of Muc, Lgr5, and mpept in WT and KO mice. The expression levels of (a) Muc, (b) Lgr5, and (c) mpept were assessed by immunofluorescence. Muc, Lgr5 and mpept were immunostained using anti-muc, anti-lgr5, and anti-pept, respectively (FITC, green). F-actin was stained using phalloidin (TRITC, red), and cell nuclei were stained using DAPI (blue). Separate pictures were taken at for each filter, and the images were merged. Scale bar, 5 µm. Supplementary Figure 3. Clustering graph of selected mirnas. MicroRNA microarray results demonstrated that different mirna profiles were ovserved in crypts and villi of WT and PepT KO mice. We selected mirnas that exhibited P value <5 and signal strengths > 5. Supplementary Figure 4. Expression levels of selected mirnas in crypt and villus epithelial cells from BL-6 WT and PepT KO mice. The expression levels of 36 selected mirnas were verified by qrt-pcr (n= 5/group; * P< 5, P< 5, and * P< ). Supplementary Figure 5. PepT expression disturbs normal differential protein expression along the crypt-villus axis. D-DIGE DeCyder BVA (Biological Variation Analysis) showing a representative gel image with labeled sample of difference. Changes in
protein expression were compared between villi and crypts, with a pooled internal standard included. The master gel image shows the locations of the differentially expressed proteins in each comparison. a. 43 spots with >.-fold differences in WT villi vs. s. b. 7 spots with >.-fold differences in KO villi vs. s. Supplementary Figure 6. PepT expression altered the normal protein profile in villus and crypt, respectively. D-DIGE DeCyder BVA (Biological Variation Analysis) showing a representative gel image with labeled sample of difference. Changes in protein expression were compared between BL-6 WT and PePT KO mice, with a pooled internal standard included. The master gel image shows the locations of the differentially expressed proteins in each comparison. a. 4 spots with >.-fold differences in WT villi vs. KO villi. b. 5 spots with >.-fold differences in s vs. s.
Supplementary Figure
a BL-6 WT Muc 5 μm 5 μm 5 μm 5 μm PepT KO Muc 5 μm 5 μm 5 μm 5 μm b BL-6 WT Lgr5 5 μm 5 μm 5 μm 5 μm PepT KO Lgr5 5 μm 5 μm 5 μm 5 μm c BL-6 WT PepT 5 μm 5 μm 5 μm 5 μm PepT KO PepT Supplementary Figure 5 μm 5 μm 5 μm 5 μm
Supplementary Figure 3
8 * mirna--5p mirna-3-3p mirna-4-5p 6 4 mirna-4-5p * 4 mirna-5-3p mirna-5a-5p 3 mirna-6-5p mirna-8a-5p. mirna-8b-5p. 3 * mirna-8a-5p mirna-9-5p mirna-94-3p Supplementary Figure 4
mirna-9b-5p 3 mirna-c-5p mirna-3-3p. mirna-a-5p mirna--3p mirna-5-5p 4 mirna--3p. mirna-3-3p mirna--3p 3 mirna-6a-5p mirna-9c-3p mirna-3c-5p *. * Supplementary Figure 4
mirna-3-3p mirna-3-5p mirna-36-3p. *.5. mirna-33-5p * mirna-345-5p.5 *. 4 3 * mirna-34a-5p 3 mirna-375-3p 4 mirna-484 3 mirna-65-3p 3 mirna-79 mirna-7-5p 8 * mirna-98-5p 6 4 * Supplementary Figure 4
MW: -~5 kda MW: -~5 kda MW: -~5 kda MW: -~5 kda a 4 WT Villus 4 WT Crypt 337 337 63 3 63 3 398 9 347 5 7 353 457 398 9 347 5 7 353 457 4 4 833 833 87 8 78 96 96 85 87 87 8 78 96 96 85 87 95 95 368 34 368 34 3 346 38 3 346 38 48 48 5 54 5 54 83 834 83 834 38 99 379 38 99 379 38 375 378 36 354 357 38 38 375 378 36 354 357 38 356 386 356 386 ph:3- ph:3- b 337 KO Villus KO Crypt 63 34 66 36 339 8 496 593 78 87 346 3 46 4 8 3 98 6 53 66 353 477 73 757 794 866 833 93 74 947 95 789 85 87 93 34 5 8 45 33 7 49 5 457 83 56 87 36 63 66 3 339 337 34 593 496 46 346 66 6 4 98 3 8 757 78 74 789 87 8 85 947 93 95 5 34 8 7 45 49 34 33 5 39 73 866 53 833 353 93 794 457 477 56 83 3 34 39 346 38 57 3 59 495 346 38 57 363 643 59 536 57 495 74 536 57 778 74 377 363 643 377 76 778 76 85 85 379 375 378 36 379 357 375 378 36 354 357 354 386 ph:3-386 ph:3- Supplementary Figure 5
MW: -~5 kda MW: -~5 kda MW: -~5 kda MW: -~5 kda a WT Villus KO Villus 37 6 937 966 993 98 37 6 937 966 993 98 6 6 398 398 4 46 794 4 46 794 593 789 593 789 87 875 87 875 47 47 54 537 54 537 38 99 38 99 354 357 38 354 357 38 386 386 ph:3- ph:3- b WT Crypt KO Crypt 77 77 875 875 7 7 537 537 ph:3-335 ph:3-335 Supplementary Figure 6
Supplementary Table. List of primers used in this study Primer Sequences 36B4 F TCCAGGCTTTGGGCATCA 36B4 R CTTTATCAGCTGCACATCACTCAGA mpept F CTCCATCTTCTACCTGTCCATCAACGCA mpept R GCTACGGTTCCTGAAGCGGTTTTTGACT mmu-mir-4-5p F CATAAAGTAGAAAGCACTACT mmu-mir-6-5p F TAGCAGCACGTAAATATTGGCG mmu-mir--3p F AAGCTGCCAGTTGAAGAACTGT mmu-mir-94-3p F CCAGTGGGGCTGCTGTTATCTG mmu-mir-33-5p F GTGCATTGTAGTTGCATTGCA mmu-mir-3-3p F TGTCAGTTTGTCAAATACCCCA mmu-mir-3-5p F TATTGCACATTACTAAGTTGCA mmu-mir-4-5p F CAGTGGTTTTACCCTATGGTAG mmu-mir-5a-5p F TAGCAGCACATAATGGTTTGTG mmu-mir-3-3p F TGCTATGCCAACATATTGCCATC mmu-mir-36-3p F CCTCTGGGCCCTTCCTCCAGT mmu-mir-9c-3p F TAGCACCATTTGAAATCGGTTA
mmu-mir-3-3p F GTGAAATGTTTAGGACCACTAG mmu-mir-9b-5p F AGTTTTGCAGGTTTGCATCCAGC mmu-mir-6a-5p F TTCAAGTAATCCAGGATAGGCT mmu-mir-3-3p F AGCAGCATTGTACAGGGCTATGA mmu-mir-9-5p F CTGACCTATGAATTGACAGCC mmu-mir-98-5p F TGAGGTAGTAAGTTGTATTGTT mmu-mir-3c-5p F TGTAAACATCCTACACTCTCAGC mmu-mir-5-5p F ATGACCTATGATTTGACAGAC mmu-mir--3p F TAACAGTCTCCAGTCACGGCCA mmu-mir--3p F AGCTACATTGTCTGCTGGGTTTC mmu-mir-a-5p F TAAAGTGCTTATAGTGCAGGTAG mmu-mir-8a-5p F AACATTCAACGCTGTCGGTGAGT mmu-mir-79 F GGAGGCAGAGGCAGGAGGA mmu-mir-484 F TCAGGCTCAGTCCCCTCCCGAT mmu-mir-8a-5p F TAAGGTGCATCTAGTGCAGATAG mmu-mir-8b-5p F AACATTCATTGCTGTCGGTGGGT mmu-mir-65-3p F AATGGCGCCACTAGGGTTGTG mmu-mir-34a-5p F TGGCAGTGTCTTAGCTGGTTGT
mmu-mir-c-5p F CGTCTTACCCAGCAGTGTTTGG mmu-mir-375-3p F TTTGTTCGTTCGGCTCGCGTGA mmu-mir-7-5p F CTCCTTCACCCGGGCGGTACC mmu-mir-345-5p F GCTGACCCCTAGTCCAGTGCTT mmu-mir-5-3p F TCAGTGCATGACAGAACTTGG mmu-mir--5p F AACCCGTAGATCCGAACTTGTG Muc F GACCTGACAATGTGCCCAGA Muc R GGCAAACACAGTCCTTGCAG Lgr5 F CTGCCCATCACACTGTCACT Lgr5 R GCAGAGGCGATGTAGGAGAC