Table S1. Primers used to quantitatively amplify the human mirnas precursors and indicated genes
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1 Table S1. Primers used to quantitatively amplify the human mirnas precursors and indicated genes Forward primer (5 3 ) Rervese primer (5 3 ) U6 CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT 5S TACGGCCATACCACCCTGAA TAACCAGGCCCGACCCTGCT let-7d AGAGGTAGTAGGTTGCATAGTT mir-101# AAAAAATACAGTACTGTGATAACTGAA mir-106a AAAAGTGCTTACAGTGCAGGTAG mir-106b# AAATAAAGTGCTGACAGTGCAGAT mir-10a TACCCTGTAGATCCGAATTTGTG mir-130b CAGTGCAATGATGAAAGGGCAT mir-142-3p# AAATGTAGTGTTTCCTACTTTATGGA mir-142-5p# AAAACATAAAGTAGAAAGCACTACT mir-146a# AAATGAGAACTGAATTCCATGGGTT mir-146b TGAGAACTGAATTCCATAGGCT mir-155 TTAATGCTAATCGTGATAGGGGT mir-15a TAGCAGCACATAATGGTTTGTG mir-16 TAGCAGCACGTAAATATTGGCG mir-17 CAAAGTGCTTACAGTGCAGGTAG mir-184 TGGACGGAGAACTGATAAGGGT mir-18a TAAGGTGCATCTAGTGCAGATAG mir-193a TGGGTCTTTGCGGGCGAGATGA mir-195 CCAATATTGGCTGTGCTGCTCC mir-198 GGTCCAGAGGGGAGATAGGTTC mir-199a* ACAGTAGTCTGCACATTGGTTA mir-19a# AAATGTGCAAATCTATGCAAAACTGA mir-19b# ATGTGCAAATCCATGCAAAACTGA mir-206 TGGAATGTAAGGAAGTGTGTGG mir-20b CAAAGTGCTCATAGTGCAGGTAG mir-21 TAGCTTATCAGACTGATGTTGA mir-214# ACAGCAGGCACAGACAGGCAG mir-22 AAGCTGCCAGTTGAAGAACTGT mir-221 AGCTACATTGTCTGCTGGGTTTC mir-222 AGCTACATCTGGCTACTGGGTC mir-23a ATCACATTGCCAGGGATTTCC mir-25 CATTGCACTTGTCTCGGTCTGA mir-26a TTCAAGTAATCCAGGATAGGCT mir-29a TAGCACCATCTGAAATCGGTTA mir-29c# ATAGCACCATTTGAAATCGGTTA mir-30a mir-30b mir-30e-3p mir-30e-5p# mir-31 mir-338 mir-342 mir-34b mir-363* mir-373* TGTAAACATCCTCGACTGGAAG TGTAAACATCCTACACTCAGCT CTTTCAGTCGGATGTTTACAGC AAATGTAAACATCCTTGACTGGA AGGCAAGATGCTGGCATAGCT AACAATATCCTGGTGCTGAGTG AGG GGT GCT ATC TGT GAT TGA CAATCACTAACTCCACTGCCAT CGG GTG GAT CAC GAT GCA ATT T ACTCAAAATGGGGGCGCTTTCC
2 mir-374a# mir-422b mir-424# mir-451# mir-523 mir-549# mir-552# mir-569# mr-656# mir-765 mir-768# mir-92 mir-93 mir-95# mir-98# Forward primer (5 3 ) Rervese primer (5 3 ) AAAATTATAATACAACCTGATAAGTG CTGGACTTGGAGTCAGAAGG AAACAGCAGCAATTCATGTTTTGAA AAAAAAACCGTTACCATTACTGAGTT GAACGCGCTTCCCTATAGAGGGT AAAAATGACAACTATGGATGAGCTCT AAAAACAGGTGACTGGTTAGACAA AAAAAGTTAATGAATCCTGGAAAGT AAAAAAAATATTATACAGTCAACCTCT TGGAGGAGAAGGAAGGTGATG GTTGGAGGATGAAAGTACGGAGTG TATTGCACTTGTCCCGGCCTG CAAAGTGCTGTTCGTGCAGGTAG AATTCAACGGGTATTTATTGAGCA AAATGAGGTAGTAAGTTGTATTGTT LV-miR-155WT GAGGATCC TAAAGGTAACAATGTCATCT GAGGTCGACGAATATATTTTCTGTTAATG LV-miR - 155MUT@ AATTACGAAATCGTGATAGGGGTTTTTGCC ACCCCTATCACGATTTCGTAATTCAGCATA pgl-kpc1-wt AACGCTAGCCTGGAACCTCCACCTTTGAA AAGCTCGAGTGTATGGAATAATAATTTAATGCTCAC pgl-kpc1-mut AACGCTAGCCTGGAACCTCCACCTTTGAA TAGCTCGAGTGTATGGAATAATAATAATTTGCTCAC pgl-cd115-wt GATCTCGAGCAAACTCTGCCTTCGGTCAT GATGTCGACTTAATGCTGTTAGTTTAATGTGGACAGA pgl-cd115-mut GATCTCGAGCAAACTCTGCCTTCGGTCAT GATGTCGACAATTACCTGTTAGTTTAATGTGGACAGA pgl-socs-1-wt AATGCTAGCCCGGCAGCGCCCGCCGTGC CATCTCGAGCTGCACAGCAGAAAAATAAAGC pgl-socs-1- MUT AATGCTAGCCCGGCAGCGCCCGCCGTGC ACGCAGCAAATTCT GAPDH GTCATCCCTGAGCTAGACGG GGGTCTTACTCCTTGGAGGC GM-CSFR GCA TTC CTC CTG ATC CCA GA CCT GGA GTC AAA CCT CAC ATT G CD115 CTAGGGACATCATGAATGACTC GTTTATAGAACTTGCTGTTCACC Note: # primers were designed by adding A at the 5 end (in bold) or truncating several bases at the 3 end to obtain a melting temperature (Tm) of C according to the manual for Ncode mirna First-Strand cdna Synthesis The seeding region sequence of mir-155 was mutated in the forward and reverse primers (in italic bold). These two primers together with primers for LV-miR-155WT were used to generate LVmiR-155MUT. Restriction enzyme sites in primers were underlined.
3 Figure S1. mirna expression analysis in human monocytes, imdcs and mdcs Shown are scatter plots of normalized mirna microarray data for monocyte versus imdc (A), monocyte versus mdc (B), and imdc versus mdc (C). Dots represent individual mirnas, and those for mir-221 and mir-155 are indicated by arrows. Figure S2. Reduced secretion of IL-12p70 by mdcs from mir-155 mice BM derived imdcs ( per well) at day 6 from WT or mir-155 (KO) mice were cultured in 24-well plates in 1 ml RPMI1640 culture medium supplemented with mgm-csf (20 ng/ml). Cells were treated with LPS (0.25 µg/ml) for 24 h. Supernatants were harvested and IL-12p70 was measured by ELISA. The data are means with 95% confidence intervals from three independent experiments. ** P<0.01 by two sample t test. Figure S3. SOCS-1 is a target of mir-155 (A) Multiple species sequence alignment of the SOCS-1 3 UTR including the predicted mir- 155 target site sequence (in bold). Mutation of the mir155 target site sequence is shown below. (B) Luciferase reporter assays to confirm SOCS-1 3 UTR targeting by mir-155. The columns represent normalized relative luciferase activity (RLU) by means with 95% confidence intervals from two independent experiments. ** P<0.01 by two sample t test. Figure S4. Over-expression of mir-155 in DCs significantly increases IL-12 production after stimulation with potent IL-12 inducers (A) Enhanced IL-12 production in DCs after over-expression of mir-155 and stimulation with LPS + R848 and poly(i:c) + the cocktail. 1,2 ImDCs at day 5 from two donors were transfected with 40 nm of mir-155 or negative control. 6 h later, cells were treated with the LPS [0.1 μg/ml, cat# L2654, Sigma] + imidazoquinoline compound R848 (2.5 µg/ml, InvivoGen) or poly(i:c) (20 µg/ml, InvivoGen) + the cocktail in AIM V serum free medium supplemented with human GM-CSF and IL-4 for 16 h. Supernatants were harvested and IL-12p70 was measured by ELISA. The data are mean ± SEM from three replicates. ** P<0.01 by two sample t test. (B) Flow cytometric analysis of CD83 maturation marker in DCs after over-expression of mir-155 and stimulation with LPS + R848 and poly(i:c) + the cocktail for 16 h. Unfilled histogram indicates an isotype control. REFERENCES 1. Kim HJ, Kim HO, Lee K, Baek EJ, Kim HS. Two-step maturation of immature DCs with proinflammatory cytokine cocktail and poly (I: C) enhances migratory and T cell stimulatory capacity. Vaccine 2010; 28(16): Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 2002; 3(2):
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Toluidin-Staining of mast cells Ear tissue was fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 C, afterwards
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Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.
Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons
c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP
Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir-484 30 20 10 0 **** **** NCP mir- 484P NCP mir- 484P b Relative
Supplementary Figure 1 a
Supplementary Figure a Normalized expression/tbp (A.U.).6... Trip-br transcripts Trans Trans Trans b..5. Trip-br Ctrl LPS Normalized expression/tbp (A.U.) c Trip-br transcripts. adipocytes.... Trans Trans
Supplementary Document
Supplementary Document 1. Supplementary Table legends 2. Supplementary Figure legends 3. Supplementary Tables 4. Supplementary Figures 5. Supplementary References 1. Supplementary Table legends Suppl.
well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±
Supplementary data: Control DC Blimp-1 ko DC 8 6 4 2-2 IL-1β p=.5 medium 8 6 4 2 IL-2 Medium p=.16 8 6 4 2 IL-6 medium p=.3 5 4 3 2 1-1 medium IL-1 n.s. 25 2 15 1 5 IL-12(p7) p=.15 5 IFNγ p=.65 4 3 2 1
Figure S1. Analysis of genomic and cdna sequences of the targeted regions in WT-KI and
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Supplemental Figures: Supplemental Figure 1 Suppl. Figure 1. BM-DC infection with H. pylori does not induce cytotoxicity and treatment of BM-DCs with H. pylori sonicate, but not heat-inactivated bacteria,
Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification.
Supplementary Table 1. Sequence of primers for real time PCR. Gene Forward primer Reverse primer S25 5 -GTG GTC CAC ACT ACT CTC TGA GTT TC-3 5 - GAC TTT CCG GCA TCC TTC TTC-3 Mafa cds 5 -CTT CAG CAA GGA
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