Table S1. Nucleotide sequences of synthesized oligonucleotides for quantitative

Transcription

1 Table S1. Nucleotide sequences of synthesized oligonucleotides for quantitative RT-PCR For CD8-1 transcript, forward primer CD8-1F 5 -TAGTAACCAGAGGCCGCAAGA-3 reverse primer CD8-1R 5 -TCTACTAAGGTGTCCCATAGCATGAT-3 TaqMan probe CD8-1T 5 -FAM-TATACACGGGCCCCATC-MGB-3 For CD8- transcript, forward primer CD8 F reverse primer CD8-R 5 -TTTTCAGGTTGTGAAACTCAACCTT-3 5 -TCCACCCGGCAGATGCTA-3 TaqMan probe CD8-T 5 -FAM-CCATTTCTGTGGACTAAT-MGB-3 For -1 transcript, forward primer Ex4-F reverse primer Ex6-R TaqMan probe Ex6/7-T 5 -GGCAGCACGGACTTGAACA-3 5 -TCCACGGAAACAGCATCTGA-3 5 -FAM-TCTTTGTGACAGTCTTGC-MGB-3 For - transcript, forward primer Ex5-F reverse primer Ex6-R TaqMan probe Ex6/7-T 5 -ACCTGGTGAGCACGGGATAC-3 same as for -1 transcript same as for -1 transcript

2 Table S. Nucleotide sequences of synthesized oligonucleotides for ChIP assay For promoter region of CD8-1 gene, forward primer -13F reverse primer -1R TaqMan probe -59T 5 -CCTAGCGCTTCCTCTTCTTTGA-3 5 -GGAATTTCCCCTTTCCAACTTC-3 5 -FAM-AGCTTCCTCCGAGGTC-MGB-3 For promoter region of CD8- gene, forward primer -659F 5 -ACCTCCTGAGTGCTGGGAATT-3 reverse primer -545R 5 -GCTTGTCTATCTTTTCTTTTTTCTTCCA -3 TaqMan probe -65T 5 -FAM-CATTCGTTTTCATTTCTC-MGB-3 For promoter region of -1 gene, forward primer -115F reverse primer -38R TaqMan probe -88T 5 -CTGAAACCGAACTCGAATAATCTACA-3 5 -TGTTCCTCTTTTCTTAGACTTGAGGAA-3 5 -FAM-AGCAAAGAAAGGGTTAAAGA-MGB-3 For promoter region of - gene, forward primer -15F reverse primer +11R TaqMan probe -6T 5 -GCCCACCTACCAGCAGAAGTTAT-3 5 -GCCCACCTACCAGCAGAAGTTAT-3 5 -FAM-TCTCTAGTCAGTTCCC-MGB-3

3 Table S3. Nucleotide sequences of synthesized oligonucleotides for cloning of promoter regions For promoter region of CD8-1 gene, forward primer CD8-pro-F1-XhoI 5 -GCCTCGAGAATCTGGAAACCATGACCTTTTGTTCAAGA-3 reverse primer CD8-pro-R1-HindIII 5 -GCAAGCTTAATCAAAAGATGGGGCCCGTGTATATACAA-3 For promoter region of CD8- gene, forward primer CD8-pro-F-XhoI 5 -GCCTCGAGCCAGTTGTAGGCATGATGTCTGTCTCCCTC-3 reverse primer CD8-pro-R-BglII 5 -GCAGATCTGGCATCCACCCGGCAGATGCTAAAGATGAT-3 For promoter region of -1 gene, forward primer -pro-f1-xhoi 5 -GCCTCGAGAGCTCTGATGGAGGGGCTCCAGCACTGCCT-3 reverse primer -pro-r1-hindiii 5 -GCAAGCTTGAGCCACTCTTACGCGTCTGCTTGCTCCTC-3 For nested PCR to amplify -1 promoter, forward primer -pro-f1-xhoi-nest 5 -GCCTCGAGGATGTTATAGACTTCTCCTCAGCAGGGTGG-3 reverse primer -pro-r1-hindiii-nest 5 -GCAAGCTTACAGGAGTCTGGTTGTTCAAGTCCGTGCTG-3

4 For promoter region of - gene forward primer -pro-f-xhoi 5 -GCCTCGAGTGGGAGTGAGACAGACGCTGCACAGAGCTT-3 reverse primer -pro-r-hindiii 5 - GCAAGCTTAAGTAAAAGGAGCCTTGTCCGCTCCCCAGG-3

5 Figure S1. Expression of CD8 and in murine BMDCs with various stimuli Murine BMDCs were stimulated with/without LPS (1 µg/ml) and/or IFNγ (1 ng/ml) for 4 hours, followed by analysis of CD8 or by flow cytometry. A, Solid line histogram represents cells with each Ab. Dotted line histogram represents negative control with.4g alone. Unstim. refers to treatment without any agents. B, Graph was generated with the relative mean fluorescence intensity (MFI) values obtained after normalization with MFI of negative control. Figure S. Schematic drawing of primer sets used for 5 -RACE (thin arrow) and for detection of transcripts in Fig. S3 (thick arrow) Figure S3. mrna expression levels of CD8 and in BMDCs mrna was prepared from murine BMDCs treated with IFNγ (1 ng/ml) for 4 hours, and/or following stimulation by LPS (1 µg/ml) for 1 hour or without any treatment. Quantification of CD8 and mrnas was performed by real-time PCR. Figure S4. Cell surface expression of CD8 and understimulation by multi-cytokine cocktails. Lineage-negative cells were transfected with pmx-ires/gfp series and transfectants were monitored as GFP-positive cells. Histogram represents cells transfected with retrovirus vector encoding PU.1 cdna or mock vector. Cells

6 were incubated with infectious viruses for 3 days in the presence of IL-3, IL-6, SCF, TPO and EPO with GM-CSF, G-CSF, M-CSF, and Flt3L. At 3 days after infection, cells were stimulated with/without 1 µg/ml LPS and/or 1 ng/ml IFNγ for 4 hours, and were then stained with PE-labeled mabs and transfectants are monitored as GFP-positive cells. Solid line histogram represents cells with each Ab. Dotted line histogram represents negative control with.4g alone. Representative results are shown. Figure S5. CD8 and sirna suppressed CD8 and expression, respectively, but did not suppress PU.1 expression. Murine BMDCs were transfected with µg of CD8 sirna, sirna, negative control sirna (N.CTRL) or distilled water (vehicle). A, After 48-hour culture, total RNA was extracted from each transfectant, and the amounts of PU.1, CD8, and β-actin mrnas were analyzed by ABI75. PU.1, CD8 and mrna levels are represented as the ratios against those of negative controls. The results are expressed as means + SD for 3 PCRs performed in duplicate. B, After 48-hour culture, cells were harvested and subjected to analysis for CD8 or by flow cytometry. Solid line histogram represents cells with each Ab. Dotted line histogram represents negative control with.4g alone. Representative results of two independent experiments are shown. C, mrna expression levels of PU.1, CD8, and in sirna-injected ears. Vehicle and negative control (n = ), PU.1 sirna (n = 4), CD8 sirna and sirna (n = 3).

7 A CD8 unstimulated FL-H FL1-H LPS FL-H FL1-H IFNγ FL-H FL1-H LPS + IFNγ FL-H FL1-H Log fluorescence intensity B 3 CD unstim.lps IFNg LPS+IFNg unstim.lps IFNg LPS+IFNg Figure S1

8 CD8 5 linker CD8-1 CD8-1T CD8- CD8-T CD8-common 3 GeneRacer 5 GeneRacer 5 nested CD8-R1-nest CD8-R1 CD8-R CD8-R3 5 linker -1-1T -1 -common 3 GeneRacer 5 GeneRacer 5 nested -R1-nest -R1 5 linker - -T - -common 3 GeneRacer 5 GeneRacer 5 nested -R1-nest -R1 Figure S

9 8 6 4 CD8-1 un LPS IFNg L+I un CD8- LPS IFNg L+I 4 CD8-common 3 1 un LPS IFNg L+I 3 1 un -1 LPS IFNg L+I un - LPS IFNg L+I 3 -common 1 un LPS IFNg L+I Figure S3

10 IL-3,IL-6,SCF,TPO,EPO + GM-CSF,G-CSF,M-CSF,Flt3L mock PU.1(WT) mock PU.1(WT) Unstim LPS IFNγ LPS,IFNγ CD8 Figure S4

11 A sirna mrna vehicle N.CTRL CD8 vehicle N.CTRL CD8 vehicle N.CTRL CD8 CD8 PU.1 B N.CTRL N.CTRL CD8-siRNA -sirna CD8 C mrna expression level * ** * ** ** sirna vehicle N.CTRL PU.1 CD8 vehicle N.CTRL PU.1 CD8 vehicle N.CTRL PU.1 CD8 mrna PU.1 CD8 Figure S5