FIGURE S1 Characterization of Mscs. A, Flow Cytometry Assay Was Used to Detect Classical MSC Marker Expression
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FIGURE S1 Characterization of MSCs. A, Flow cytometry assay was used to detect classical MSC marker expression. Passage: P4. B, MSCs were induced to osteocytes by incubation with osteogenic differentiation medium (RASMX-90021, Cyagen) and stained by alizarin red. C, MSCs were induced to adipocytes by incubation with adipogenic differentiation medium (RASMX-90031, Cyagen) and stained by Oil red O. D, MSCs were induced to chondrocytes by incubation with chondrogenic differentiation medium (RASMX-90041, Cyagen) and stained by HE. Bar: 100 μm FIGURE S2 Effect of miR-503 on A549 cells in vitro. A, XTT assay was used to detect cell proliferation in A549 cells transfected with miR-503 mimics or miR-503 AS, or their respective controls at 24 hours post-transfection. B, Flow cytometry assay was used to detect cell proliferation in A549 cells transfected with miR-503 mimics or miR-503 AS, or their respective controls at 24 hours post-transfection. Apoptosis inducer (Cisplatin, 1 μM, Beyotime) were added to the culture. All data are shown as the mean ± standard deviation based on three independent experiments, ** p<0.01, * p<0.05. C, Matrigel assay was used to detect the formation of capillary-like tubes in A549 cells transfected with miR-503 mimics or miR-503-AS, or their respective controls at 24 hours post-transfection. Bar: 100 μm. miR-nc AS: antisense strands for miR-nc; miR-503 AS: antisense strands for miR-503; FIGURE S3 miRNAs differentially expressed in rMSCs exposed to TCM, compared with control rMSCs. A, Heatmap showed the differentially expressed miRNAs in rMSCs exposed to TCM, compared with control rMSCs, Each small block indicates its own expression level. Red denotes a high expression level, green denotes a low expression level. B, List of miRNAs differentially expressed in rMSCs exposed to TCM, compared with control rMSCs. Supplementary Table 1. Oligonucleotide sequences for siRNA and target genes. Name Sequence miRNA miR-503 Sense 5’-uagcagcgggaacaguacugcag-3’ Antisense 5’-gcaguacuguucccgcugcuauu-3’ miR-NC Sense 5’-uucuccgaacgugucacgutt-3’ Antisense 5’-acgugacacguucggagaatt-3’ miR-503 AS 5’-cugcaguacuguucccgcugcua-3’ miR-nc AS 5’-uuguacuacacaaaaguacug-3’ Primers miR-503 Stem-loop 5’-gtcgtatccagtgcgtgtcgtggagtcggcaattgcactggatacgacctgcag-3’ Sense 5’- tagcagcgggaacagta -3’ Antisense 5’- tgcgtgtcgtggagtc -3’ u6 Sense 5’-gcttcggcagcacatatactaaaat-3’ Antisense 5’-cgcttcacgaatttgcgtgtcat-3’ α-SMA Sense 5’-aactggtattgtgctggactctg-3’ Antisense 5’-cccatcaggcagttcgtagc-3’ FAP Sense 5’-atgaagacatggctgaaaaccg-3’ Antisense 5’-ggctgtaaactcttgagggacg-3’ FSP Sense 5’-gaaggtgttgagcgggaagaa -3’ Antisense 5’-gagacgagaaggagcattggtt-3’ TNC Sense 5’-gctttgtttgccctcactcct-3’ Antisense 5’-caccactggctgattctcctct -3’ CCL20 Sense 5’-gcagccagtcagaagcagcaa-3’ Antisense 5’-ccaattccatcccagaaaagc-3’ IL1Β Sense 5’-acctgctagtgtgtgatgttccca -3’ Antisense 5’-aggtggagagctttcagctcacat-3’ CXCL2 Sense 5’-caatgcctgacgaccctacc-3’ Antisense 5’-ttttggacgatcctctgaacc-3’ CXCL3 Sense 5’-ccttcagggactgttgtggc-3’ Antisense 5’-ggctatgacttctgtctgggtg-3’ VEGF Sense 5’-cgaaaccatgaactttctgc -3’ Antisense 5’-cctcagtgggcacacactcc-3’ SDF-1 Sense 5’-tgcccatgccgattcttc-3’ Antisense 5’-ggctttgcccaggttgact-3’ IGF-1 Sense 5’-ggtggatgctcttcagttcgt-3’ Antisense 5’-cactccctctacttgcgttctt-3’ MMP-3 Sense 5’-ttcctggcatcccgaagt-3’ Antisense 5’-ggtccatcaaaagggtaaaagtc-3’ β-ACTIN Sense 5’-tggaatcctgtggcatccatgaaac -3’ (RAT) Antisense 5’-taaaacgcagctcagtaacagtccg -3’ GAPDH Sense 5’-gaccactttgtcaagctcatttcc-3’ (HUMAN) Antisense 5’-gtgagggtctctctcttcctcttgt-3’ Table S2. List of genes differentially expressed in MSCs exposed to TCM, compared with control MSCs Genes Up-regulat OLR1142, CCL20, HTR3B, IL1B, LOC683206, CXCL2, CXCL3, ion LOC100910497, CXCL1, ASS1, CCL7, CCL2, PLA2G2A, DRD1, (fold >1.5) MMP3, RSAD2, CCL4, CCL3, C3, HP, CXCL10, SLPI, RTP4, RBP1, CMPK2, TNIP3, OAS1K, MGC105649, GCH1, TYRP1, SLFN2, TLR2, RAC2, LOC100910885, IFIH1, NFKBIA, ISG15, OLR1695, MMP9, CCL12, TGM2, TMEM106A, ZC3H12A, HPN, CREG1, ANGPTL4, BDKRB1, CXCL14, LCN2, THBS4, CD83, NFKBIZ, TNF, IL18BP, C1S, ZC3HAV1, BCL3, ADORA2B, G0S2, RGD1305725, RGD1559629, FMOD, HSD11B1, VCAM1, LOC360228, CXCL13, PF4, EPN3, ACER1, PTGS1, CXCL16, CX3CL1, TRH, FCGR2B, FCGR1A, LOC24906, KRT12, CCL5, APOL9A, GDF15, IL1A, LOC498276, LRP1B, CYP7B1, ABCB1B, ANGPT4, SOD2, PTGS2, IRF7, CD40, TSPAN11, MT1A, NFKBIE, CP, BIRC3, RBM38, SLC25A37, PLK2, HDAC11, C1R, MAP3K8, DDX58, ENPP2, LOC654482, CYP4A8, STAP2, MT2A, GADD45A, SERPING1, ADM2, MMP12, CCR2, LOC678766, RAB32, RGD1311756, TUBA8, KCNT1, RAP1GAP2, NTN1, ICAM1, GJB2, TMEM86A, CAMP, CYP26B1, BMP6, DIP2C, USP18, GPRC5A, CD200, NCF1, IFI204, FLI1, ARHGDIB, KLF10, RGD1559864, RASL12, N4BP2L1, EGR2, RHBDF2, TCP11, SELP, ALDH3B1, SERINC2, HCK, KCNQ5, ABCG1, RGD1561149, CPXM1, ATOH7, RASD1, SLAMF9, CDC42EP3, CSF1, RGD1564086, RT1-CE2, DIO3, TAP2, OLR1, DNAJC12, GINS2, PADI3, LOC100362769, DAGLA, CEBPB, ITPKA, PLA1A, RT1-A2, RT1-CE16, TGS1, VEGFA, GRAMD1B, ZFP174, TNFRSF1B, GGT1, TFPI2, TMEM144, RGD1305587, POPDC2, LYZ2, IRF1, UPP1, RT1.AA, GBP5, LOC361914, F3, GREM1, LAMB3, SLC43A2, KLHL29, RPRD1B, CCRN4L, METRN, FSCN2, SPINT2, PSMB9, BOD1, OLR1616, PARVB, TNFSF15, PPP1R15A, RBPJ, CD14, EDN1, PHLDA1, ECHDC3, OPTN, EGR3, SLC35F2, CYP4F17, ZFP512B, LOC684871, TXNIP, EVA1C, BMP2, FAM110A, N4BP3, AGRP, STAC2, TSLP, FCER1G, SHROOM1, EML2, SLC9B2, ARSE, CCDC90A, EFNB1, WNT11, FOXS1, PHLDB3, SLC2A1, PTPDC1, DUSP16, TFRC, PPIP5K1, EMP2, LCN6, TMEM140, P2RX4, SMOC2, CLU, MAFK, ZFP133, LOC498752, WDSUB1, MOGAT2, LAMA5, MICALL2, BLOC1S6, ZMIZ1, ANXA3, MOSPD1, GLUL, IFI44, CST6, MGC125239, LOC687813, CPT1A, LOC100912739, MMACHC, NAGLU, CLCN3, HOGA1, MGMT, AKNA, SH2B2, CSRNP1, PVRL1, PMAIP1, ADAMTSL5, SLC20A1, SAMD4A, GDNF, TRAF2, DMRT2, HOOK2, CAMK1G, RT1-T18, KCNQ4, FAM206A, KLF5, BCL6, FAM5B, CDK17, LTBP2, GBP2, SLC38A7, LPCAT2, KDSR, LOXL4, VEGP2, LOC682105, SLC16A11, RGD1562079, KLK14, SCUBE3, NR1H3, PANX1, RDH12, ZNRF1, ARHGEF6, RGD1304982, RSPH9, SLC29A1, PKNOX1, B3GNT7, DFNB31, IER3, HTR6, DSN1, IRAK3, C8G, GAS7, RGD1566380, ENO2, MSH5, PLAT, UPK1B, PTX3, CORO1A, SMIM3, ABCA2, ANGEL1, LMNB1, ETV6, PYGL, TRIB3, WNT5A, TBC1D2, ISG20L2, SLC11A2, RNASE1, KLHL30, LOC100910973, SMG9, ITGA10, PLAGL2, EMR1, NMRAL1, ATF3, TNFAIP2, DPEP1, SLC3A2, TMEM132E, RGD1560281, ACOT2, CYP1A1, SPP1, HSPA2, RGD1566373, IFIT2, RAMP3, ATG16L2, TK2, CPEB1, RT1-A1, SH3BP2, PHLDA3, LOC690483, FBXO10, CHIC2, CYP2S1, LIF, SPRY2, NFKBIB, EEPD1, CD55, TRAF3, PDE10A, JUNB, CARS, SHISA8, RGD1304595, RTN4R, LOC100359980, RT1-S2, KLHL41, GPM6B, DUSP5, GIPC2, NCEH1, RENBP, MLLT4, AIF1, RAB36, CITED2, CRLF1, TRIO, BDH2, CYP4F5, TLE3, BLCAP, ALKBH7, GPD2, TNKS1BP1, PSEN2, PRKAB2, ENPP4, TRIT1, S1PR1, HNRNPUL2, PIM3, CCDC28B, CIDEA, BHLHE40, NUDT2, LOC691666, NR1D1, KLF15, ETS2, AGXT2L2, PLAC8, SIK1, LOC687696, UBE2L6, CYS1, CCDC8, DUSP14, SLC7A3, FUOM, FLCN, CDKN2AIPNL, WDR12, KLHDC8A, WDTC1, USP54, PLEKHG4, SLC7A5, PIGV, CCDC28A, UNK, HEMK1, KCNMA1, TNFRSF22, TNIP1, AMBRA1, MT3, DHRS3, MANBA, AGTR1A, NFKB2, HES1, SMURF1, LOC684681, RT1-S3, RAD9B, GMFB, RGD1564114, PARP14, RASL11B, LHFPL2, LEF1, LOC499770, STOML1, ACSF3, RGD735029, MAP2K5, SLC51B, GOT1, CTH, UAP1L1, ZC3H8, RT1-M6-1, CACNA1G, PLA2G6, RT1-CE7, PEX3, RT1-CE5, MAML1, TGFB2, MRI1, IL11RA1, GFER, STEAP3, BCL2, RASSF2, NCOA2, GRHL3, DPH2, UBALD2, BCAR1, PAOX, MDM2, PDE2A, SUN2, TIMP3, SERPINE1, RGD1304624, SPERT, YDJC, NFKB1, IFNGR2, ACOT8, HINT3, PROSC, PRND, CXCL12, CCDC32, SURF2, MAPKAPK3, LOC307263, PKMYT1, RWDD3, SAT2, RAB20, SDC4, CCDC19, ING3, CYP1B1, TAF8, ROPN1L, EXTL3, GINM1, SLC7A1, PRDX5, EVI2A, MGST1, GPNMB, PARP8, NMNAT3, IFI27L2B, AMDHD2, LOC501224, RT1-CE14, NRG1, FAM131B, PNRC1, DNASE2, RGD1309708, LOC100125367, CCBE1, MCOLN1, LGALS3, LYRM9, PEX6, RT1-CL, TYROBP, TMEM218, APOL3, GCAT, BCL2L1, ACADS, PROCR, DDX59, TFIP11, MIB2, YARS, LOC100911534, OTUB2, LOC684270, CYM, LOC685438, ZXDB, FAM49A, TMEM63A, PCED1A Down- MORC1, CSAD, IGSF11, OLR1266, HS3ST2, TMCO5A, PKM, regulation ZFP334, SHISA7, ITPR2, POU2AF1, VOM2R24, CCDC117, TREM3, SPTA1, IDI1, SPEM1, SCD1, CYP51, HMGCS1, RGD1561594, (fold >1.5) PCSK9, ASB15, RGD1560208, FDPS, ZC4H2, MEPE, ASPN, CDH6, FBXW8, INSIG1, EDNRA, DEPDC1, ELOVL6, SH3PXD2A, MEST, TRIL, PPARD, LOC689246, HSD17B7, MVD, H19, BLNK, DHCR7, PANX3, SQLE, COL6A3, OLR305, FADS1, POLH, ADAMTSL3, HMGCR, GZMB, ACAT3, NOX4, MALL, NEXN, FAM189A2, STARD4, FRMPD1, GAS2, SCD, PRELID2, OLR545, PROM1, RGD1305422, HTR4, MMP15, CADPS2, PCDH19, ERG, TCEAL8, VOM2R68, MSMO1, ADRA1D, GLS, ELN, OGN, MBOAT1, E2F7, NEFL, HYLS1, APOBEC2, CSTF1, NPAS2, FASN, ROCK1, PLN, PRPF39, VSNL1, SLC18A2, UHMK1, OLR857, SOX15, C1QL3, IGSF10, PARK2, ARSK, AMH, PLS3, SLC29A2, ITCH, ACSS2, C1QTNF3, SLC7A14, ANO1, ARHGAP11A, TMEM97, HLTF, THSD7A, AKAP17B, ASPM, MLC1, SOWAHA, VCSA1, ITM2A, UBE2QL1, OLR745, HTR2A, CDR2, PTH1R, SEMA3D, ABP10, SESN3, SERPINA11, LOC683788, TLR13, LRRC17, PCMTD2, BIRC6, OMD, CALCB, VAV3, FAM81A, ADA, MRVI1, CLOCK, RPL37, FAM13A, ETAA1, APOBEC1, PRM1, MIER1, PCOLCE2, SENP5, OLR416, USP9X, LOC691717, RGD1305110, HMGN5, LOC304131, UBD, LOC691984, DCLK1, SHANK3, SYNJ2BP, PNN, LOC494539, TPH1, RGD1563084, COL2A1, PBRM1, TYMP, SLC22A8, HOXC5, SRSF7, OLFML2A, GPER, FRAS1, KCTD2, PRRT1, FBN2, NMNAT1, INPP5F, OLR1057, ABCG2, OLR674, GUCY1B3, CD163L1, MYH1, PCDHA13, SEC23A, NOTCH1, FOXD4, RPF2, IPO7, AOC3, GAPDH-PS1, FUT8, DNM1L, NSDHL, RGD1563520, TNKS2, MAGEB7, GLT8D1, ZFP386, PSME4, TM7SF2, PCDH7, RGL1, ST6GALNAC3, PTGER3, SLC5A5, POLR3G, TDRD3, VCAN, VPS13B, TTC3, IBSP, MS4A15, NOXO1, CBFB, TUBA1A, TSC22D2, EIF5, DDX3X, GPR182, COBL, CCDC88A, SMARCA1,