Analysis of Mir-34A-5P Over-Expression by Qrt-PCR

Supplementary material files

Supplementary Figure 1: Analysis of miR-34a-5p over-expression by qRT-PCR.

Primary CD4+ T cells were transfected either with allstars negative control (ANC) or miR-34a-5p mimic. 48 h post transfection the total RNA was isolated and analyzed by qRT-PCR using a specific hsa-miR-34a-5p primer.

Supplementary Figure 2: FACS Controls

CD4+ T cells were stained for CD4 and co-stained for CD11A or the respective isotype controls for 30 min at 4°C. Cells were analyzed by flow cytometry. Gated

CD4+ T cells (A) were analyzed for CD11A expression (B, dark grey) in comparison to isotype controls (light grey).

CD8+ T cells were stained for CD8 and co-stained for CD11A or the respective isotype controls for 30 min at 4°C. Cells were analyzed by flow cytometry. Gated

CD8+ T cells (C) were analyzed for CD11A expression (D, dark grey) in comparison to isotype controls (light grey).

Supplementary Table 1

target cloning sequence ID gene primers (restriction sites are underlined) 5'-VAMP2-SpeI ggactagtccatcatcctcatcatcatc VAMP2 NM_014232.2 3'-VAMP2-NaeI gccggccctcaatcagttcacccaatgag 5'-IKBKE-SpeI ggactagtcacatgaggcatcctgaag IKBKE NM_014002.3 3'-IKBKE-SacI cgagctcgcatagaaagaacaggaggctc 5'-MYH9-SpeI ggactagtcgaagaggtagatggcaaagc MYH9 NM_002473.5 3'-MYH9-SacI cgagctccctttgtgacagcaactggg 5'-MARCH8-SpeI ggactagtgtgtgcgggttgtcattttc MARCH8 NM_001282866.1 3'-MARCH8-SacI cgagctccagaatgcactgagagtggg 5'-KLRK1-SpeI ggactagtgagactgtgcactctatgcctc KLRK1 NM_007360.3 3'-KLRK1-SacI cgagctcccttcttaactgtgaacctgtg 5'-CD11A-SpeI ggactagtgagaaggactctgagagtgg CD11A NM_002209.2 3'-CD11A-SacI cgagctcctctaactcctgacctcaggtg 5'-TRAFD1-SpeI ggactagtcatcggttcctgtcttctgtg TRAFD1 NM_001143906.1 3'-TRAFD1-SacI cgagctcctgacacttggttttcagagc 5'-CCR1-SpeI ggactagtctgactcagaccataggagg CCR1 NM_001295.2 3'-CCR1-NaeI gccggccctcctacatcaacacagaatc 5'-PYDC1-SpeI ggactagtgacatgcgcatgttggag PYDC1 NM_152901.3 3'-PYDC1-SacI cgagctcccatgaatatttattgggcagtggaagaataaatgacag 5'-PRF1-SpeI ggactagtgaacagtgagcttggaaaggg PRF1 NM_001083116.1 3'-PRF1-SacI cgagctccaaagaagacagagcagctg 5'-PIK3R2-SpeI ggactagtctgtctctctccatgttggg PIK3R2 NM_005027.3 3'-PIK3R2-SacI cgagctccaacgtaccgtacaaaacctg 5'-PIK3CD-SpeI ggactagtgaccaagcacattggtcc PIK3CD NM_001350234.1 3'-PIK3CD-SacI cgagctcggtttcacagagtgctggtc 5'-AP1B1-SpeI ggactagtcatcctcaagaactgagacc AP1B1 NM_001127.3 3'-AP1B1-SacI cgagctcgatctagttaccgtagggtc 5'-ADAM10-SpeI ggactagtgtgttccctgttcttcgttg ADAM10 NM_001110.3 3'-ADAM10-SacI cgagctcccacatcttgattcgcagtgg 5'-PVR-SpeI ggactagtgaccccaatggatgaagac PVR NM_006505.4 3'-PVR-SacI cgagctcgtgggttatttcctgccagtc 5'-AP1B1-SpeI ggactagtgaaacagctgctgatgctac AP2S1 NM_001301076.1 3'-AP1B1-SacI cgagctcgacggcaaggccaggcaggaccacaggtttattgg 5'-BAD-SpeI gactagtcagctggacgcgagtcttcc BAD NM_004322.3 3'-BAD-SacI cgagctccatttggtagtgagcacg 5'-ICOS-SpeI ggactagtcatgagagcagtgaacacag ICOS NM_012092.3 3'-ICOS-SacI cgagctccggtatagtctgacagcaacc 5'-CD247-SpeI ggactagtctacctgctggatggaatcc CD247 NM_198053.2 3'-CD247-NaeI gccggcggtcagtctgttcatcttctgg 5'-ZPF36-SpeI ggactagtcgcatctctgtttctgagtg ZPF36 NM_003407.3 3'-ZPF36-SacI cgagctcccagcttacactcagattg 5'-STX8-SpeI ggactagtcagaaagtcagcctcttgtg STX8 NM_004853.2 3'-STX8-SacI cgagctccagagctttgggggaatttattgagag 5'-SPN-SpeI ggactagtgtgtcggtgaatagtgaggc SPN NM_001030288.2 3'-SPN-SacI cgagctccttcccagactctggtcaac Supplementary Table 2: predicted miR-34a target genes

predicted targets in immune system process DLL1 PRKCQ CACNB3 ARIH2 EFNB1 ESRRA KLRK1 HOXB4 MYH9 SERPINE1 CALCR ADAM10 CDK6 CALCOCO2 NDRG1 LYST CRHR1 PVR DPP4 SWAP70 CXCL10 KIT KITLG NFKBIA NUB1 PKNOX1 PAG1 PURB PVRL1 CCL22 SNCA VCAM1 ZFP36 CAV1 TOB2 FSTL3 CEACAM8 TRAFD1 GPR44 CHRNB2 RASGRP4 CCR1 ANKRD54 CRP CX3CR1 VAMP2 CYP11B1 predicted targets in immune system process CFD EDN2 RTKN2 ICOSLG DDX58 PYDC1 GBP2 GCH1 B4GALT1 POLL HLA-DQA1 HLX HMGB1 TLX1 TMEM173 AP1B1 IFI35 IKBKB IL1R1 AQP3 IRAK2 ITPKB KCNJ8 ARHGDIB MARCH8 LTF MITF ATM OAS3 GLRX5 PDGFB PML STX8 TOLLIP PRKCA CXCL16 RFX1 BCL6 SMAP1 ROCK1 CXCL11 CXCL5 PRDM16 SELPLG SPN TAL1 TGFBR2 TGFBR3 TLR4 predicted targets in immune system process WNT2B TFEB ROGDI ICOS NLRC5 TN- FRSF14 BCL10 CCND3 MAP3K14 RSAD2 IL1RL1 CD247 GPR55 CD28 CXCL14 GTPBP1 CD47 CD97 P2RY14 RC3H1 PIK3CD SARM1 IRF1 JARID2 SP2 TACR1 WNT1 BCL2 TRIM10 CPLX2 AP2S1 ADD2 ADK CR2 NFAM1 IL28RA POLR3H FKBP1B JMJD6 IL1RN PIK3R2 IL6R INPP5D KIR2DL1 KIR3DL1 CD46 NOTCH4 FOXP3 predicted targets in immune system process ENPP1 PLCG1 SOX6 CTNNBIP1 BAD SATB1 TCF7 THRA C1QC UNG VEGFA XBP1 PRF1 CBFA2T3 ACVR1B CD3E CD86 CD44 ABCC9 SEMA4D MS4A2 SIT1 IRF4 ITGAL JAK2 LY9 LY75 SMAD3 MLL OSM MASP1 ST6GAL1 BMP6 TCF12 TPD52 CALCA ULBP2 IKBKE SH2B3 PATZ1 MAPK1 NOTCH2 PODXL ITCH JAG1 NOTCH1 BCL11B SOX4 Supplementary Table 3: validated miR-34a target genes

miRTarBase validated Target Support References miRNA Experiments ID Gene Type (PMID)

Luciferase reporter hsa-miR- Functional MIRT000074 JAG1 assay//qRT- 20351093 34a-5p MTI PCR//Western blot

Flow//Immunobl ot//Luciferase hsa-miR- Functional MIRT000074 JAG1 reporter 19398721 34a-5p MTI assay//Microarr ay//qRT-PCR

Flow//In situ hybridization//L uciferase hsa-miR- reporter Functional MIRT000074 JAG1 24307639 34a-5p assay//Microarr MTI ay//qRT- PCR//Western blot

Non- hsa-miR- MIRT001010 CCND3 Western blot Functional 18406353 34a-5p MTI

qRT- PCR//Luciferas hsa-miR- e reporter Functional MIRT001012 CDK6 18406353 34a-5p assay//Western MTI blot//Reporter assay

hsa-miR- Luciferase Functional MIRT001012 CDK6 19461653 34a-5p reporter assay MTI qRT- hsa-miR- PCR//Western Functional MIRT001012 CDK6 19584398 34a-5p blot//Luciferase MTI reporter assay miRTarBase validated Target Support References miRNA Experiments ID Gene Type (PMID)

Luciferase hsa-miR- reporter Functional MIRT001012 CDK6 17914404 34a-5p assay//Western MTI blot//Microarray

Luciferase reporter hsa-miR- Functional MIRT001012 CDK6 assay//qRT- 19773441 34a-5p MTI PCR//Western blot hsa-miR- Western Functional MIRT001012 CDK6 21240262 34a-5p blot;Other MTI qRT- hsa-miR- Functional MIRT001012 CDK6 PCR//Western 23035210 34a-5p MTI blot Functional hsa-miR- MIRT001012 CDK6 PAR-CLIP MTI 23592263 34a-5p (Weak) hsa-miR- Luciferase Functional MIRT001231 WNT1 19336450 34a-5p reporter assay MTI Flow//Immunobl ot//Luciferase hsa-miR- Functional MIRT001231 WNT1 reporter 19398721 34a-5p MTI assay//Microarr ay//qRT-PCR Luciferase reporter hsa-miR- Functional MIRT001231 WNT1 assay//qRT- 28199987 34a-5p MTI PCR//Western blot hsa-miR- Luciferase Functional MIRT001757 DLL1 14697198 34a-5p reporter assay MTI hsa-miR- Luciferase Functional MIRT001757 DLL1 19461653 34a-5p reporter assay MTI hsa-miR- Luciferase Functional MIRT001757 DLL1 20144220 34a-5p reporter assay MTI Luciferase reporter hsa-miR- Functional MIRT001757 DLL1 assay//qRT- 23327670 34a-5p MTI PCR//Western blot miRTarBase validated Target Support References miRNA Experiments ID Gene Type (PMID) Luciferase reporter hsa-miR- Functional MIRT002298 BCL2 assay//qRT- 18803879 34a-5p MTI PCR//Western blot Luciferase reporter assay//qRT- hsa-miR- PCR//Western Functional MIRT002298 BCL2 19683563 34a-5p blot//Reporter MTI assay;Western blot;qRT- PCR;Other qRT- hsa-miR- PCR//Western Functional MIRT002298 BCL2 18505919 34a-5p blot//Luciferase MTI reporter assay hsa-miR- Luciferase Functional MIRT002298 BCL2 19461653 34a-5p reporter assay MTI

Luciferase hsa-miR- reporter Functional MIRT002298 BCL2 17914404 34a-5p assay//Western MTI blot//Microarray

Luciferase reporter hsa-miR- Functional MIRT002298 BCL2 assay//Microarr 17656095 34a-5p MTI ay//Western blot Flow//Immunobl ot//Luciferase hsa-miR- Functional MIRT002298 BCL2 reporter 20598588 34a-5p MTI assay//Reporter assay hsa-miR- Reporter Functional MIRT002298 BCL2 21399894 34a-5p assay;Other MTI Functional hsa-miR- MIRT002298 BCL2 Proteomics MTI 21566225 34a-5p (Weak) qRT- hsa-miR- Functional MIRT002298 BCL2 PCR//Western 24565525 34a-5p MTI blot hsa-miR- Luciferase Functional MIRT002298 BCL2 23862748 34a-5p reporter assay MTI miRTarBase validated Target Support References miRNA Experiments ID Gene Type (PMID) Functional hsa-miR- MIRT002298 BCL2 qRT-PCR MTI 23292172 34a-5p (Weak) Functional hsa-miR- MIRT002298 BCL2 qRT-PCR MTI 26802970 34a-5p (Weak) qRT- hsa-miR- Functional MIRT002298 BCL2 PCR//Western 27939626 34a-5p MTI blot hsa-miR- Flow//QRTPCR Functional MIRT002298 BCL2 26406332 34a-5p //Western blot MTI Luciferase reporter hsa-miR- Functional MIRT002298 BCL2 assay//qRT- 25910896 34a-5p MTI PCR//Western blot hsa-miR- Luciferase Functional MIRT002875 NOTCH1 14697198 34a-5p reporter assay MTI Functional hsa-miR- MIRT002875 NOTCH1 Microarray MTI 19461653 34a-5p (Weak) Luciferase reporter hsa-miR- Functional MIRT002875 NOTCH1 assay//qRT- 20351093 34a-5p MTI PCR//Western blot Luciferase reporter hsa-miR- Functional MIRT002875 NOTCH1 assay//qRT- 19773441 34a-5p MTI PCR//Western blot Luciferase hsa-miR- reporter Functional MIRT002875 NOTCH1 17150773 34a-5p assay//Microarr MTI ay Immunofluores cence//Immuno histochemistry// Luciferase hsa-miR- Functional MIRT002875 NOTCH1 reporter 22363487 34a-5p MTI assay//Microarr ay//qRT- PCR//Western blot miRTarBase validated Target Support References miRNA Experiments ID Gene Type (PMID) Luciferase reporter hsa-miR- Functional MIRT002875 NOTCH1 assay//qRT- 22684561 34a-5p MTI PCR//Western blot hsa-miR- Luciferase Functional MIRT002875 NOTCH1 23085450 34a-5p reporter assay MTI hsa-miR- Functional MIRT002875 NOTCH1 Western blot 23226240 34a-5p MTI hsa-miR- Microarray//We Functional MIRT002875 NOTCH1 22438124 34a-5p stern blot MTI qRT- hsa-miR- Functional MIRT002875 NOTCH1 PCR//Western 23035210 34a-5p MTI blot qRT- hsa-miR- Functional MIRT002875 NOTCH1 PCR//Western 23430952 34a-5p MTI blot Luciferase hsa-miR- reporter Functional MIRT002875 NOTCH1 28145431 34a-5p assay//Western MTI blot Luciferase reporter hsa-miR- Functional MIRT002875 NOTCH1 assay//qRT- 26493107 34a-5p MTI PCR//Western blot ELISA//Flow//L uciferase hsa-miR- reporter Functional MIRT002875 NOTCH1 28129650 34a-5p assay//qRT- MTI PCR//Western blot ELISA//Lucifera Non- hsa-miR- MIRT004513 VEGFA se reporter Functional 18320040 34a-5p assay MTI Luciferase reporter hsa-miR- Functional MIRT005029 NOTCH2 assay//qRT- 19773441 34a-5p MTI PCR//Western blot miRTarBase validated Target Support References miRNA Experiments ID Gene Type (PMID) Immunoblot//Im munocytochemi stry//Luciferase hsa-miR- reporter Functional MIRT005029 NOTCH2 22363487 34a-5p assay//Microarr MTI ay//qRT- PCR//Western blot Immunohistoch emistry//qRT- hsa-miR- Functional MIRT005480 CD44 PCR//Western 21240262 34a-5p MTI blot//Reporter assay;Other Luciferase reporter hsa-miR- Functional MIRT005480 CD44 assay//qRT- 24423412 34a-5p MTI PCR//Western blot qRT- hsa-miR- Functional MIRT005480 CD44 PCR//Western 25572695 34a-5p MTI blot Flow//qRT- hsa-miR- Functional MIRT005480 CD44 PCR//Western 27497057 34a-5p MTI blot qRT- hsa-miR- Functional MIRT005480 CD44 PCR//Western 26231042 34a-5p MTI blot Luciferase reporter hsa-miR- Functional MIRT005480 CD44 assay//qRT- 25551284 34a-5p MTI PCR//Western blot Flow//Luciferas hsa-miR- e reporter Functional MIRT006286 ULBP2 22102694 34a-5p assay//Western MTI blot Functional hsa-miR- MIRT006286 ULBP2 PAR-CLIP MTI 26701625 34a-5p (Weak) hsa-miR- Luciferase Functional MIRT006869 CCL22 22975373 34a-5p reporter assay MTI Functional hsa-miR- MIRT025289 EFNB1 Proteomics MTI 21566225 34a-5p (Weak) miRTarBase validated Target Support References miRNA Experiments ID Gene Type (PMID) Functional hsa-miR- MIRT025289 EFNB1 PAR-CLIP MTI 23592263 34a-5p (Weak) Functional hsa-miR- MIRT025369 PLCG1 Proteomics MTI 21566225 34a-5p (Weak) Functional hsa-miR- MIRT025409 NDRG1 Proteomics MTI 21566225 34a-5p (Weak) Functional hsa-miR- MIRT025584 TPD52 Sequencing MTI 20371350 34a-5p (Weak) Functional hsa-miR- MIRT025584 TPD52 Proteomics MTI 21566225 34a-5p (Weak) Functional hsa-miR- MIRT307050 TGFBR2 PAR-CLIP MTI 22012620 34a-5p (Weak) Western hsa-miR- Functional MIRT437939 ARHGDIB blot//Luciferase 23349340 34a-5p MTI reporter assay// Luciferase hsa-miR- reporter Functional MIRT438239 KIT 24009080 34a-5p assay//Western MTI blot hsa-miR- Luciferase Functional MIRT438239 KIT 27056900 34a-5p reporter assay MTI ChIP- seq//Immunoflu hsa-miR- Functional MIRT438704 IL6R orescence//qRT- 24642471 34a-5p MTI PCR//Western blot Luciferase reporter hsa-miR- Functional MIRT438704 IL6R assay//qRT- 25973020 34a-5p MTI PCR//Western blot hsa-miR- Luciferase Functional MIRT438704 IL6R 26091352 34a-5p reporter assay MTI Functional hsa-miR- MIRT535098 PODXL PAR-CLIP MTI 22012620 34a-5p (Weak) miRTarBase validated Target Support References miRNA Experiments ID Gene Type (PMID) Functional hsa-miR- MIRT702573 JARID2 HITS-CLIP MTI 23313552 34a-5p (Weak) qRT- PCR//Luciferas hsa-miR- e reporter Functional MIRT731117 TCF7 25436980 34a-5p assay//Immuno MTI histochemistry// Western blot Immunocytoche mistry//Lucifera hsa-miR- Functional MIRT732534 CACNB3 se reporter 25623948 34a-5p MTI assay//Western blot Functional hsa-miR- MIRT732593 MAP3K14 qRT-PCR MTI 25864118 34a-5p (Weak) Luciferase reporter hsa-miR- Functional MIRT733801 NLRC5 assay//qRT- 27423514 34a-5p MTI PCR//Western blot Functional hsa-miR- MIRT733971 BCL10 PCR array MTI 28097098 34a-5p (Weak) hsa-miR- Luciferase Functional MIRT734565 FKBP1B 26471303 34a-5p reporter assay MTI Functional hsa-miR- MIRT741440 POLL PAR-CLIP MTI 26701625 34a-5p (Weak) miR-34a target genes validated by Hart et al. Luciferase hsa-miR- reporter Functional n.a. PRKCQ 27144431 34a-5p assay//Western MTI blot Luciferase hsa-miR- reporter Functional n.a. PRKCA 27144431 34a-5p assay//Western MTI blot Luciferase hsa-miR- reporter Functional n.a. NFKBIA 30718475 34a-5p assay//Western MTI blot Luciferase hsa-miR- Functional n.a. CD3E reporter 30718475 34a-5p MTI assay//FACS