-. -. SUPPLEMENTARY INFORMATION DOI: 1.1/ncb86 a WAT-1 WAT- BAT-1 BAT- sk-muscle-1 sk-muscle- mir-133b mir-133a mir-6 mir-378 mir-1 mir-85 mir-378 mir-6a mir-18 mir-133a mir- mir- mir-341 mir-196a mir-17 mir-434-3p mir- mir-99 mir-486 mir-361 mir- mir-155 mir-379 mir-181b mir-14 mir-185 mir-15b-5p mir-199a-3p mir-181a mir-199a-5p mir-76 mir-14 mir-13 mir-17 mir-1a mir-78 mir-19 mir-148a mir-3d mir-93 mir-451 mir-191 mir-3 mir-35 mir-15 mir-45 let-7i mir-146a mir-34-3p mir-99b mir- mir-1b mir-34a mir-195 mir-8 mir-18 mir-15a-5p mir-139-5p mir-15b mir-1 mir-335-5p mir-146b mir-13a mir-4 mir-143 mir-1 mir-99a mir-1 mir-c mir-b mir-151-5p mir-18 mir-455 mir-16 mir-16-3p mir-3c mir-16b mir-3 mir-9a mir-3a mir-3 mir-15 let-7g mir-a mir-193 mir-6b mir-15a mir-17 mir-9b b c Non-RT control d P1 mir-193b mir-365 13448k 134k 1345k 13451k 1345k 13453k 13454k 13455k P mir-193b P3 P4 P1 P P3 P4 P5 P6 P7 P8 P9 P1 P P3 P4 f 15 1 5 P5 P5 P6 Wt ob/ob P6 e P7 P7 P8 P9 4.kb P8 P9 P1 Basal 4 O C P11 P1 mir-365-1 P13 P14 P1 P11 P1 P13 P14 P1 P11 P1 P13 Basal camp 5 4 3 1 Dio P14 log(sample/mean) Supplemental Fig 1 Figure S1 MiR-193b-365 is a brown fat-enriched mirna cluster. (a). Detailed heat map showing the expression levels of 91 mirnas that are expressed in at least one sample at an intensity > 5 arbitrary units and are differentially expressed between the three tissues (P <.1, ANOVA). Red denotes higher and green denotes lower level relative to the mean of the six samples for each mirna. Tissues from 4-5 mice were pooled for each RNA sample preparation. (b). Cap-analysis gene expression (CAGE) Basic and Analysis Databases store original results produced by CAGE-seq, which measures expression levels of transcription start sites by sequencing large numbers of 5 transcript ends, termed CAGE tags. CAGE tag distribution across the genomic region around -365 is shown here. A black arrow represents a single 5 transcript tag, and a red arrow represents a cluster of 5 transcript tags. There is no CAGE tag between and -1 in the direction of transcription, supporting the idea that and are co-transcribed. (c). RT- PCR analysis of the primary transcript of -365. Random primers were used to generate cdna from primary brown adipocytes. 14 pairs of primers (P1 to P14) depicted in the diagram above were then used to amplify segments of the putative primary transcript that includes both and. The amplified fragments covered the entire region between and, indicating that -365 is a bona fide cotranscribed mirna cluster. (d). Real-time PCR analysis of and mir- 365 expression in BAT of wild- type and ob/ob mice (1 week old). n=5. (e). 8 week old male mice were exposed to 4 o C for 4 hrs to induce thermogenesis in BAT. RNAs of interscapular BAT were harvested and real-time PCR was performed to examine the expression of and, and two markers genes upregulated during thermogenesis, type II iodothyronine deiodinase (Dio) and uncoupling protein 1 (). n=5. (f). Brown fat SVF cells were cultured and differentiated to day 6 and stimulated with 5uM dibutyrul camp for 4h. The expression of, and was examined by RT-PCR. n=3. P < 5, Student s t-test; Means ± SEM. WWW.NATURE.COM/NATURECELLBIOLOGY 1 11 Macmillan Publishers Limited. All rights reserved.
SUPPLEMENTARY INFORMATION a 1 5 d 1μm LNA-miR-1 LNA- LNA-miR-193a+b FITC - CD45 1 4 1 3 LNA-miR-1 LNA-miR-193a LNA- LNA-miR-193a+b+365 1 3 5 15 1 5 b 65.8% 1 1 3 1 4 1 5 APC - CD11b 87.9% 1 1 3 1 4 1 5 PE - Sca1 LNA mir-193a+b LNA 6 8 e f g.. LNA- Gapdh Cebpa mir-1 LNA- LNA-+365 8 Pgc-1a Pparg Prdm16 LNA- LNA- LNA- LNA- LNA-+365 LNA-+365 c Relative Expression Relative Expression Cidea LNA-193a+b LNA-365 Adipogenesis markers AdipoQ Dio CebpA Pgc1a Brown fat markers Ppara Pparg Prdm16 i h LNA-miR-193a+b LNA- Fig S AdipoQ Cebpa LNA-miR-193a+b LNA- 5μm Glut4 Pparg Figure S MiR-193b-365 is required for brown fat adipogenesis. (a). Brown fat SVF cells were FACS-sorted to enrich brown preadipocyte population (CD45 -, CD11b -, Sca1 + ). Sorted cells were transfected with LNA inhibitors for mir-193a+b and, then induced to differentiation for 4 day. (b). ORO staining and (c). Real time-pcr were used to examine the effects of blocking these mirnas on brown fat adipogenesis. n=3. (d). SVF cells were transfected with LNA mirna inhibitors (1nM) one day before differentiation. 4 days after differentiation, representative micrographs were taken and are shown here. (e). Western blot to examine the expression of in response to blocking and/or during brown fat adipogenesis. (f, g). Primary brown preadipocytes were cultured and differentiated to day 6. These cells were transfected by LNA mirna inhibitors as indicated in figure. 3 days after, RNAs were extracted and RT-PCR was performed to examine the expression of (f) mirnas and (g) brown fat markers. (h). Subcutaneous white fat SVF cells were transfected with LNA mirna inhibitors for mir-193a+b and, and then were induced to differentiation for 4 day. ORO staining and Real time-pcr (i) were used to examine the effects of blocking these mirnas on white fat adipogenesis. n=3. P < 5, Student s t-test; Means ± SEM. WWW.NATURE.COM/NATURECELLBIOLOGY 11 Macmillan Publishers Limited. All rights reserved.
SUPPLEMENTARY INFORMATION c e a 1 8 6 4 D D6 Runx1t1-P1 Runx1t1-P1 Runx1t1-P Runx1t1-P b GFP Runx1T1 d Runx1t1 AdipoQ 5μm Adipogenesis markers Cebpα Pparγ Runx1t1 Cidea Dio Brown fat markers Pgc1α Pparα Prdm16 f LNA-miR-193a+b LNA-miR-193a+b Runx1t1 6 46 Gapdh 8 Figure S3 MiR-193b regulates adipogenesis by targeting Runx1t1. (a). Real-time PCR to examine the change of Run1t1 during brown fat adipogenesis. (b-e). Brown fat SVF cells were infected by retrovius expressing RUNX1T1, and then differentiated for 4 days. (b) ORO staining and (c-e) Real-time PCR were performed to examine the effects of ectopic Runx1t1 on brown fat adipogenesis. n=3. (f). Brown fat SVF cells were transfected with LNA mirna inhibitors for mir-193a and mir- 193b and then were induced to differentiate. Western was performed to examine the level of Runx1t1. P < 5, Student s t-test; Means ± SEM. WWW.NATURE.COM/NATURECELLBIOLOGY 3 11 Macmillan Publishers Limited. All rights reserved.
SUPPLEMENTARY INFORMATION a 8 6 4 4 3 1 Primary brown adipocytes Primary brown adipocytes b mir-193a AACUGGCCUACAAAGUCCCAGU AACUGGCCCACAAAGUCCCGCU mir-193b mir-365-1 c mir-13 d mir-193a CC1 mir-17 Myf5 Myf6 MyoG Ckm Tnni Tnnt3 Casq1 e +365 188 f 188 98 6 8 Myosin GAPDH 98 6 8 Cdon Gapdh Igfbp5 Figure S4 MiR-193b-365 represses myogenesis of CC1. (a). Realtime- PCR analysis of and in differentiated CC1 myoblasts retrovirally expressing these two mirnas. Primary brown adipocytes directly isolated from animals were used as a positive control. Ectopic expression resulted in a mirna level about ~3 and ~6.5-fold higher than in primary brown adipocytes for and, respectively, indicating that the mirna expression levels were in a physiological range. n=3. (b). Diagram to show the nucleotide sequences of mir-193a (upper), and mir- 193b (lower) and their relative positions in the genome. (c). Representative micrographs of cells (Day 6) differentiated from CC1 myoblasts expressing retroviral mir-193a or the control vector. (d). Real-time PCR analysis of cells depicted in (c) for their expression of muscle markers, Myf5, Myf6, Myog, muscle creatine kinase [Ckm], troponin I type [Tnni], troponin T type 3 [Tnnt3], calsequestrin 1 [Casq1]. n=3. (e). Western blot with triplicate biological repeats to examine the expression of myosin upon ectopic expression of and/or. n=3. (f). Western analysis for predicted targets, cell adhesion molecule-related/down-regulated by oncogenes (Cdon) and insulin-like growth factor binding protein 5 (Igfbp5), in CC1 cells expressing or a control vector. n=3. P < 5, Student s t-test; Means ± SEM. 4 WWW.NATURE.COM/NATURECELLBIOLOGY 11 Macmillan Publishers Limited. All rights reserved.
SUPPLEMENTARY INFORMATION a 1 1 8 3 1 Pparγ +365 Cebpα b AdipoQ BAT_D6 CC1 D5 Cebpα Pparγ c 188 98 6 8 Pax3 188 98 6 8 MyoD 188 98 6 8 188 98 6 Gapdh 8 d f 5μm Myogenic condition Ckm Myf5 Myf6 Myod Myog Tnnt3 Tnni e Adipogenic condition g 8 6 4 AdipoQ Cebpa Ckm Myf5 Myf6 Myod Myog Tnni Tnnt3 Pparg Lpl Pgc1a Figure S5 induces adipogenesis in myoblasts. (a). CC1 cells were infected with retrovirus expressing mir-193 and/or and then exposed to myogenic condition for 6 days. Real-time PCR analysis to examine the expression of Pparγ and Cebpα. n=3. (b). CC1 myoblasts were infected by retrovirus and exposed to proadipogenic differentiation conditions for 5 days. RNAs were harvested, and Real-time PCR was used to compare the levels of Pparg, Cebpa, AdipoQ and in these cells and their levels in primary brown adipocyte cultures. Values of BAT cells are set at. n=3, (c). Western blot for myogenic markers Pax3, MyoD and brown fat marker. n=3. (d-g). Primary human myoblasts were infected with virus expressing or vector and then exposed to myogenic condition (d, e) or adipogenic condition (f, g) for 6 days. Representative micrographs of cells were shown in (d). Realtime PCR was performed to examine the expression of myogenic markers (e, f) and adipogenic markers (g). n=3. P < 5, Student s t-test; Means ± SEM. WWW.NATURE.COM/NATURECELLBIOLOGY 5 11 Macmillan Publishers Limited. All rights reserved.
SUPPLEMENTARY INFORMATION a b c 5 15 1 5 5 4 3 1 d 15 1 5 WAT Cox5b CC1 Pparγ Cidea Pparα 3 1 Pparγ WAT CC1 Myf5 Myod Cebpα Myog mir-193b mir-365 f Amplicon 1345k 13451k 1345k 13453k 13454k 15 1 5 WAT Amplicon Amplicon BAT sh- m e g 15 1 5 CC1 Amplicon sinc sippara Amplicon i k l h j Retroviral AdipoQ Amplicon WT PPARα KO Day (-3) Day (-1) Day () Day (3) LNA mir-193a+b LNA Adipogenesis markers Cebpa Inhibitor Pparg Brown fat markers Adipogenic induction Cidea Dio Ppara Prdm16 ORO staining RNA extraction LNA mir-193a+b LNA LNA mir-193a+b LNA PPARα -365 Runx1t1 Myf5+ Cdon Igfbp5 Brown adipocytes Figure S6 Figure S6-365 is regulated by. (a) Real-time PCR analysis of differentiated primary white adipocytes and (b) differentiated CC1 myoblasts that retrovirally express. White preadipocytes and CC1 myoblasts were cultured to about 5% confluence and infected by retroviruses expressing Prdm16. Six days after differentiation, cells were harvested and analyzed by real-time PCR. n=3. P < 5. (c). RT-PCR primers were designed to amplify four fragments in the primary transcript between and. (d). Subcutaneous white pre-adipocytes and (e). CC1 myoblasts were infected by retrovirus expressing Prdm16 two days before differentiation. 5 days after differentiation, real-time PCR were performed to examine the expression of pri--365. n=3. (f), Primary brown preadipocytes were infected by retrovirus expressing shrna targeting two days before differentiation. By D5 of differentiation, real-time PCR was used to examine the mrna level of pri--365. n=3. (g), Muscle Brown fat SVF cells were transfected with sirnas targeting PPARα, and differentiated for 3 days. RT-PCR was performed to examine the expression of pri- -365. n=3. (h), RT-PCR was performed to examine the expression of pri--365 in brown adipose tissue isolated from PPARα knockout mice (8 week old, Male). Age-matched wild- type mice were used as control. n=6. (i). The diagram of experimental design for (i-l). (j). ORO staining and (k, l) Real-time PCR were performed to examine the effects of LNA mirna inhibitors on brown fat adipogenesis in the presence of ectopic Prdm16. (m) Brown adipocytes arise from a Myf5-positive, myoblastic lineage. During this process, Prdm16 is strongly induced. Probably through interactions with Ppara, Prdm16 promotes the expression of the mir- 193b-365 cluster, which is required for brown fat adipogenesis. At the same time, -365 blocks the otherwise myogenic potential of brown preadipocytes. P < 5, Student s t-test; Means ± SEM. 6 WWW.NATURE.COM/NATURECELLBIOLOGY 11 Macmillan Publishers Limited. All rights reserved.
Supplementary Table 1. Real-time PCR primers FORWARD REVERSE 18S GTAACCCGTTGAACCCCATT CCATCCAATCGGTAGTAGC AdipoQ CGATTGTCAGTGGATCTGACG CAACAGTAGCATCCTGAGCCCT Casq1 GGGGGCTCATGGTAGAGGAG CTGGCACTGCTGTTTGTACTG Cdon ATCCAGACCTCGGACCCTTAT GGCAGTAACAGGTTTAGCAGAAC Cebpa TGCGCAAGAGCCGAGATAAA CCTTCTGTTGCGTCTCCACG Cidea TGCTCTTCTGTATCGCCCAGT GCCGTGTTAAGGAATCTGCTG Ckm GCAAGCACCCCAAGTTTGA ACCTGTGCCGCGCTTCT Cox8b GAACCATGAAGCCAACGACT GCGAAGTTCACAGTGGTTCC Dio CAGTGTGGTGCACGTCTCCAATC TGAACCAAAGTTGACCACCAG Elovl3 TCCGCGTTCTCATGTAGGTCT GGACCTGATGCAACCCTATGA Eva1 GTCCCAACCAGACCATCAAC CTCCATCTTGCTCTGGAAGC ACAAGCTGGTGGTGGAATGTG CCTTTGGCTCATGCCCTTT Glut4 CTGTCGCTGGTTTCTCCAACT CCCATAGCATCCGCAACATA Igfbp5 CCCTGCGACGAGAAAGCTC GCTCTTTTCGTTGAGGCAAACC Myf5 AAGGCTCCTGTATCCCCTCAC TGACCTTCTTCAGGCGTCTAC Myf6 ATCAGCTACATTGAGCGTCTACA CCTGGAATGATCCGAAACACTTG MyoD CGCCACTCCGGGACATAG GAAGTCGTCTGCTGTCTCAAAGG MyoG AGCGCAGGCTCAAGAAAGTGAATG CTGTAGGCGCTCAATGTACTGGAT Nrip1 GGCTGTCTGATTCCATCGTGA CCAGCAATGTGCTATCCTGTT Pgc1a CCCTGCCATTGTTAAGACC TGCTGCTGTTCCTGTTTTC Ppara AGAGCCCCATCTGTCCTCTC ACTGGTAGTCTGCAAAACCAAA Pparg GTGCCAGTTTCGATCCGTAGA GGCCAGCATCGTGTAGATGA Prdm16 CAGCACGGTGAAGCCATTC GCGTGCATCCGCTTGTG Retnla TCCACTCTGGATCTCCCAAGA TCCCTCCACTGTAACGAAGACTC Tnni AGAGTGTGATGCTCCAGATAGC AGCAACGTCGATCTTCGCA Tnnt3 GGAACGCCAGAACAGATTGG TGGAGGACAGAGCCTTTTTCTT 11 Macmillan Publishers Limited. All rights reserved.
ACTGCCACACCTCCAGTCATT CTTTGCCTCACTCAGGATTGG Casq1 (human) CACCCAAGTCAGGGGTACAG GTGCCAGCACCTCATACTTCT myog (human) GCTGTATGAGACATCCCCCTA CGACTTCCTCTTACACACCTTAC CEBPA (human) ACTGCGATGGGGGTCACCAG CTCCGGTCCCTCTGGGATGG FABP4 (human) AGCACCATAACCTTAGATGGGG CGTGGAAGTGACGCCTTTCA Tnni (human) ATCTGCGGGGCAAGTTCAAG AGGACTCGGACTCAAACATCT CKM (human) ATGCCATTCGGTAACACCCAC TCTTGTAGAGTTCAAGGGTCAGT PPARg (human) CCTATTGACCCAGAAAGCGATT CATTACGGAGAGATCCACGGA MyoD1 (human) CTTTGCCACAACGGACGACT GTGCTCTTCGGGTTTCAGGAG Myf6 (human) GGAGCGCCATCAGCTATATTG ATCCGCACCCTCAAGATTTTC Myf5 (human) GTCCCCCGAGGGTGAATTTG GCCCACATGAGGCAGTGAC LPL (human) TCATTCCCGGAGTAGCAGAGT GGCCACAAGTTTTGGCACC Tnnt3 (human) TCCAGAAGAAGCGTCAGAACA TCTCTGCACGAATCCTCTGCT PGC1 (human) CCAGGAGGCAGAAGAGCCGT GCTCCGAGCAGGGACGTCTT Runx1t1-P1 ATGCCTGATCGTACCGAGAAG GTCGTTGGCGTAAATGAGCTG Runx1t1-P ATTTACGCCAACGACATTAACGA CTGAGTTGCCTAGCACCACA Pri--365 Pri--365 Amplicon Pri--365 Pri--365 AGGGAGACGGTTGCAGGGTA GCTGGCTGTCACATGATAA GCTTCCCTCCCCTGCCAATA TCACTCCGTGCCAGTCCAGA TGTGACAGCCAGCAACTCAGC GGGAAGGATAACTGAGTGTCT CCTTTCCTAGGTAAGACTCCACCAC AGCACTGATGCCAACAGGCC 11 Macmillan Publishers Limited. All rights reserved.
Supplementary Table. Cloning primers Name Forward Reverse mmu- mir- 193 mmu- mir- 193b mmu- mir- 365 CDON -3UTR IGFBP 5-3UTR CDON - 3UTR- Mut IGFBP 5-3UTR- Mut RUNX 1T1-3UTR RUNX 1T1_ Mut ccgctcgagccccgtcgtgtaaaccttcga G ccgctcgagcccctacagcaggaagaggt AGG ccgctcgagcccgttgctgtacagatgctg AG CCGCTCGAGGAGGAGTGTGAGCAGA CCT CCGCTCGAGCAGTTGGCTGGGAAGG AA TCTTCGTGTTCCCACTGCAAGGATCC AACTGTACCACACAGGCCTGG CCCAACTCAAACTTCAATATATCTTAG GGATCCATCCTAGACCTAAGTCTCTC CTTTCTGC CCGCTCGAGCTGTCAGAGGAAAGACA CCAC TGAGGCGATAGTAGAACACAGAGGGA TCCAACGGGTCGTAATGACTTACTGT G ggaattcttaattaaaaactttccagtcgcttc TATCC ggaattcttaattaaaaacttccctttgacgga ATGAG ggaattcttaattaaaaatattcaccactggct TTGAG ATAAGAATGCGGCCGCTCCCCTGTTTT CCAGATGA ATAAGAATGCGGCCGCTGTGGCTCAC AGCGAAGA CCAGGCCTGTGTGGTACAGTTGGATC CTTGCAGTGGGAACACGAAGA GCAGAAAGGAGAGACTTAGGTCTAGG ATGGATCCCTAAGATATATTGAAGTTTG AGTTGGG ATAAGAATGCGGCCGCTTAGCATTTTT GCTTTCAACAG CACAGTAAGTCATTACGACCCGTTGGA TCCCTCTGTGTTCTACTATCGCCTCA 11 Macmillan Publishers Limited. All rights reserved.
Supplementary Table 3. Primers to detect the co-transcript of -365 mmu--365 transcript; P1-F mmu--365 transcript; P1-R mmu--365 transcript; P-F mmu--365 transcript; P-R mmu--365 transcript; P3-F mmu--365 transcript; P3-R mmu--365 transcript; P4-F mmu--365 transcript; P4-R mmu--365 transcript; P5-F mmu--365 transcript; P5-R mmu--365 transcript; P6-F mmu--365 transcript; P6-R mmu--365 transcript; P7-F mmu--365 transcript; P7-R mmu--365 transcript; P8-F mmu--365 transcript; P8-R mmu--365 transcript; P9-F mmu--365 transcript; P9-R mmu--365 transcript; P1-F mmu--365 transcript; P1-R mmu--365 transcript; P11-F mmu--365 transcript; P11-R mmu--365 transcript; P1-F mmu--365 transcript; P1-R mmu--365 transcript; P13-F mmu--365 transcript; P13-R CGAGAGGGGAGGTAGCG AGTAAGCTGCTTCAGACTCTCG CAGCTTACTACCTTCCCCTAA CAATCCAGATTGGGAGAA TTCACGTCTTCTGAGGACACATCC AGGGAGGTTCATCTGCACCC GAACCTCCCTACAGCAGGAAGAG CGGAGCTAGATAAGAATACCCTCCC GTGCCAAATTCTCGGAACAAAC TGTCTGTTGTCATTCAGGTGACTGA CACACACATTCCATATATGCACCC GCTCGTTAATTCACAGAAAGCACTC GCCAGATCCCTCTACTCTGCTAGA TATTGGCAGGGGAGGGAAGC GCTTCCCTCCCCTGCCAATA CCTTTCCTAGGTAAGACTCCACCAC GAAAGGCCCTGGGTTCATCC TGGGAAGGGACCACAAAGCG TCACTCCGTGCCAGTCCAGA AGCACTGATGCCAACAGGCC TGCTCAAGACAGCCTGGACC TGCTTCTAGGCCCATGGCTT AGGGAGACGGTTGCAGGGTA TGTGACAGCCAGCAACTCAGC GCTGGCTGTCACATGATAA GGGAAGGATAACTGAGTGTCT 11 Macmillan Publishers Limited. All rights reserved.
mmu--365 transcript; P14-F mmu--365 transcript; P14-R TCCTTCCCTGAGGCTTCACC AACCAATCAGCTGGCGGGAG 11 Macmillan Publishers Limited. All rights reserved.
Supplementary Table 4. ChIP primers ChIP 193b 1F ChIP 193b 1R ChIP 193b F ChIP 193b R ChIP 193b 3F ChIP 193b 3R GTGTGCTTGCAGCGGTGTCC CCCCCAAAGCCAGGGAGTCA GGTGGATGGGGTGGGGTGTT CCCCTCTGGGGTTCGCAGTT GTTAGGGGACCCCCTCCCTC GCACATTCCTCCGCTCCCCT 11 Macmillan Publishers Limited. All rights reserved.