Supplementary Information. MicroRNA-33b knock-in mice for an intron of sterol regulatory

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Supplementary Information MicroRNA-33b knock-in mice for an intron of sterol regulatory element-binding factor 1 (Srebf1) exhibit reduced HDL-C in vivo Takahiro Horie, Tomohiro Nishino, Osamu Baba, Yasuhide Kuwabara, Tetsushi Nakao, Masataka Nishiga, Shunsuke Usami, Masayasu Izuhara, Fumiko Nakazeki, Yuya Ide, Satoshi Koyama, Naoya Sowa, Naoya Yahagi, Hitoshi Shimano, Tomoyuki Nakamura, Koji Hasegawa, Noriaki Kume, Masayuki Yokode, Toru Kita, Takeshi Kimura, and Koh Ono

Supplementary Table S1 Serum lipid profiling of WT and mir-33b KI +/+ mice by HPLC Lipoprotein Major (Fraction No.) Diameter Sub-class (Fraction No.) WT (n=4) KI +/+ (n=5) TC (mg/dl) 72.71 ± 5.42 52.56 ± 3.91* CM (1-2) >8nm.73 ±.14.36 ± 6* VLDL (3-7) 3-8nm 3.93 ±.22 3.76 ±.41 LDL (8-13) 16-3nm 1.86 ±.6 9.85 ±.78 large LDL (8) 3.1 ± 9 3.39 ±.24 medium LDL (9) 2.85 ±.11 3.15 ±.21 small LDL (1) 1.77 ±.14 1.63 ±.18 very small LDL (11-13) 2.73 ±.6 1.68 ±.26 HDL (14-2) 8-16nm 57.2 ± 4.92 38.59 ± 3.23* very large HDL (14-15) 2.3 ±.68 ±.14* large HDL (16) 18.49 ± 2.44 8.89 ± 1.38** medium HDL (17) 24.9 ± 1.46 18.97 ± 1.29* small HDL (18) 8.45 ±.43 7.74 ±.47 very small HDL (19-2) 3.5 ±.25 2.32 ±.25 TG (mg/dl) 22.4 ± 2.48 19.27 ± 2.66 Values are mean ± SEM. Blood was obtained from chow-fed 8 weeks old male mice, after 4 h fasting. The serum was analyzed by HPLC, as described in material and methods. *p<5, **p<1 compared with WT mice. TC, total cholesterol; TG, triglyceride; CM, chylomicrons; VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein.

a Human SREBF1 SREBF2 SREBP-1 mir-33b SREBP-2 mir-33a Mouse Srebf1 Srebf2 SREBP-1 SREBP-2 mir-33(a) mir-33a; GUGCAUUGUAGUUGCAUUGCA mir-33b; GUGCAUUGCUGUUGCAUUGC b 133bp 24bp 166bp 475bp 21bp 89bp 112bp Human SREBF1 Ex.15 Ex.16 mir-33b Ex.17 Ex.18 133bp 168bp 166bp 41bp 21bp 98bp 112bp Mouse Srebf1 Ex.15 Ex.16 Ex.17 Ex.18 Exon 16 1 GAGAGACCCCTGCCCAGGGCAGCTCTGCACTCCTTCAAGGCTGCCCGGGCCCTGCTGGGC 6 1 GAGAGACCCCTGCCCAGGGCAGCTCTGTACTCCTTCAAGGCTGCCCGGGCTCTGCTGGAC 6 61 TGTGCCAAGGCAGAGTCTGGTCCAGCCAGCCTGACCATCTGTGAGAAGGCCAGTGGGTAC 12 61 CACAGAAAGGTGGAATCTAGCCCAGCCAGCCTGGCCATCTGTGAGAAGGCCAGTGGGTAC 12 Exon 17 121 CTGCAGGACAGCCTGGCTACCACACCAGCCAGCAGCTCCATTGACAAGGCCGTGCAGCTG 18 121 CTGCGGGACAGCTTAGCCTCTACACCAACTGGCAGTTCCATTGACAAGGCCATGCAGCTG 18 181 TTCCTGTGTGACCTGCTTCTTGTGGTGCGCACCAGCCTGTGGCGGCAGCAGCAGCCCCCG 24 181 CTCCTGTGTGATCTACTTCTTGTGGCCCGTACCAGTCTGTGGCAGCGGCAGCAGTCACCA 24 241 GCCCCGGCCCCAGCAGCCCAGGGCACCAGCAGCAGGCCCCAGGCTTCCGCCCTTGAGCTG 3 241 GCTTCAGTCCAGGTAGCTCACGGTACCAGCAATGGACCCCAGGCCTCTGCTCTGGAGCTG 3 31 CGTGGCTTCCAACGGGACCTGAGCAGCCTGAGGCGGCTGGCACAGAGCTTCCGGCCCGCC 36 31 CGTGGTTTCCAACATGACCTGAGCAGCCTGCGGCGGTTGGCACAGAGCTTCCGGCCTGCT 36 Exon 18 361 ATGCGGAGG 369 361 ATGAGGAGG 369 Supplementary Figure S1. Scheme of SREBFs and mir-33s and homologies in exons 16 and 17 of SREBF1 between human and mouse. (a) mir-33b is lacking in intron of mouse Srebf1. Mature sequences of mir-33a and mir-33b are indicated in the inset. (b) There are high homologies in exons 16 and 17 between human SREBF1 and mouse Srebf1 (82.6% of nucleotides and 79.7% of amino acids).

a WT ES clones 5 probe 35.2 kb (WT allele) 19.6 kb (Targeted allele) 1.7 kb (WT allele) 3 probe 4.8 kb (Targeted allele) Neo probe 6.6 kb b mir-33a (A.U.) 2.5 2. * *** ES clone for injection c Srebf1 (A.U.) 2. d Srebf2 (A.U.) e f Srebf1 (A.U.) mir-33b (A.U.) Brain Heart Liver Kidney Sub WAT Epi WAT Gastrocnemius Brain Heart Liver Kidney Sub WAT Epi WAT Gastrocnemius Supplementary Figure S2. Generation of mir-33b knock-in mice. (a) Southern blotting analysis of genomic DNA from ES cells that were successfully targeted by a KI vector. Representative images are shown. (b-d) Quantitative real-time PCR analysis of mir-33a (b), Srebf1 (c) and Srebf2 (d) in the liver of 8-wk-old WT, KI +/- and KI +/+ male mice (n=6). Values are mean ± s.e.m. *p < 5 ***p < 1 by one-way analysis of variance. (e, f) Quantitative real-time PCR analysis of Srebf1 (e) and mir-33b (f) in 8-wk-old KI +/+ male mice (n=3). Values are mean ± s.e.m. Sub WAT indicates subcutaneous white adipose tissue. Epi WAT indicates epididymal white adipose tissue.

a b c d mir-33b (A.U.) mir-33a (A.U.) Srebf1 (A.U.) Srebf2 (A.U.) e 15 f 15 ABCA1 (%) 1 5 * ABCG1 (%) 1 5 * Supplementary Figure S3. The expression levels of mir-33s and Srebfs in KI macrophages. (a-d) Quantitative real-time PCR analysis of mir-33b (a), mir-33a (b), Srebf1 (c) and Srebf2 (d) in peritoneal macrophages from 8-wk-old WT and KI +/+ male mice (n=3). Values are mean ± s.e.m. N.D., not determined. (e, f) Densitomeric analysis of ABCA1 (e) and ABCG1 (f) in peritoneal macrophages from 8-wk-old WT and KI +/+ male mice. (n = 4). Values are mean ± s.e.m. *p < 5 by Student s t-test.

a b c Liver weight (g) 2. ** Liver weight (g) 2. ** Srebf1 (A.U.) 3 2 1 ** Vehicle T91317 Vehicle T91317 Vehicle T91317 Supplementary Figure S4. Liver weight and Srebf1 expression of T91317-treated mice. (a) Liver weight of vehicle or T91317-treated 8-wk-old KI +/+ male mice. Values are the means ± s.e.m. (n = 6, **p<1 by Student s t-test). (b) Liver weight of vehicle or T91317-treated 8-wk-old WT male mice (n=4, **p<1 by Student s t-test). (c) Quantitative real-time PCR analysis of Srebf1 in the liver of vehicle or T91317-treated 8-wk-old WT male mice (n=4, **p< 1 by Student s t-test).

a b c ABCA1 (%) 15 1 5 ** Mature SREBP-1 (%) 15 1 *** * 5 SRC1 (%) 2 15 1 5 * d e f 2 15 15 PCK1 (%) 15 1 5 CREB (%) 1 5 G6Pase (%) 1 5 Supplementary Figure S5. The expression levels of cholesterol and glucose metabolic genes. The protein expression levels of ABCA1 (a), SREBP-1 (b), SRC1 (c), PCK1 (d), CREB (e) and G6Pase (f) in the liver of 8-wk-old WT, KI +/- and KI +/+ male mice by densitometry. The expression levels were normalized with β-actin or TF2B expressions. The expression levels of WT mice were set at 1%. Values are mean ± s.e.m. *p < 5, **p<1 ***p < 1 compared with WT by one-way analysis of variance (a-d; n=6, e and f; n=3).

Insulin LXR ligand SREBF1c mir-33b LXRE SRE SREBF1c mrna mir-33b SRE SREBP-1 responsive gene FASN, ACC, SCD1 Supplementary Figure S6. Proposed feedback mechanism to regulate SREBP-1 level by intronic mir-33b.

2b 35.2 kb 19.6 kb 1.7 kb 4.8 kb 6.6 kb 2c 3 bp 2d 3 bp 1 bp 5 bp 2 bp 1 bp 5 bp 4a ABCA1 Precursor SREBP-1 2 kd 116 kd 97 kd 4b SRC1 2 kd 116 kd short exposure 116 kd 97 kd PCK1 CREB 36 kd 31 kd Mature SREBP-1 long exposure β-actin 42 kd CPT1a 97 kd G6Pase 36 kd 31 kd β-actin 42 kd AMPKα 97 kd 5a TF2B 36 kd 2 kd 42 kd 116 kd β-actin 42 kd ABCA1 ABCG1 β-actin S2a 35.2 kb 19.6 kb 1.7 kb 6.6 kb 4.8 kb Supplementary Figure S7. Scans of the original blots in figure 2b, 2c, 2d, 4a, 4b, 5a and S2a.

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