Yue Wang, Zhenyu Xu, Junfeng Jiang, Chen Xu, Jiuhong Kang, Lei Xiao, Minjuan Wu, Jun Xiong, Xiaocan Guo, and Houqi Liu

Transcription

1 Developmental Cell, Volume 25 Supplemental Information Endogenous mirna Sponge lincrna-ror Regulates Oct4, Nanog, and Sox2 in Human Embryonic Stem Cell Self-Renewal Yue Wang, Zhenyu Xu, Junfeng Jiang, Chen Xu, Jiuhong Kang, Lei Xiao, Minjuan Wu, Jun Xiong, Xiaocan Guo, and Houqi Liu Inventory of Supplementary Information Figure S1 related to Figure 1 (A) OCT4 or NANOG knockdown results in the decrease of linc-ror in self-renewal hesc X-01. (B) The kinetic expression levels of linc-ror and core TFs in differentiated X-01 hesc. Figure S2 related to Figure 2 (A, B) Transient transfection efficiency of linc-ror overexpressing vector, control vector, linc-ror-specific sirna and control RNA. (C-F) Relative mrna and protein levels of OCT4, SOX2, and NANOG in linc-ror-overexpressing or knockdown hes X-01 cells. (G) Effort of linc-ror on the Oct4 promotor reporter in hescs. Figure S3 related to Figure 3 (A)The kinetic expression levels of mirnas in differentiated hescs. (B)RIP analysis for the Ago2 protein.

2 (C) mirna expression profiling upon knockdown and overexpressin of linc-ror and Oct4 knockdown. (D) The influence of mir-145 to linc-ror expression levels. (E-G) The target validation using luciferase reporters in self-renewal hescs H1. Figure S4 related to Figure 4 (A-B) linc-ror suppresses mature mir-145 expression in hesc X-01 line. (C) Copy numbers of linc-ror and mir-145 in two hesc lines under self-renewal or differentiated state. Figure S5 related to Figure 5 (A-B, D-E) The role of linc-ror in the maintenance of core TFs and hesc X-01 self-renewal. (C) The apoptosis rates in linc-ror knock-down hesc H1 and X-01. Figure S6 related to Figure 6 Characteristic of linc-ror-overexpressing cells (A-D) and the kinetic expression levels of core TFs and mir-145 in linc-ror knock-down cells (E). Table S1 realted to Figure 3 The predicted mirnas targeting OCT4, NANOG, SOX2 or linc-ror. Supplemental experimental procedures

3 Supplementary Information Supplemental Figures and Legends Figure S1 related to Figure 1 Linc-RoR expression is positively correlated with the undifferentiated state of ES cell X-01. (A) The relative level of linc-ror decreased in self-renewal hesc X-01 3 days after the transfection of sirnas (si) targeting OCT4 or NANOG. The interfering efficiency was confirmed with qrt-pcr. Data are represented as mean ± SEM. **, p<0.01, n=3. (B) The kinetic expression levels of linc-ror, NANOG, SOX2, and OCT4 in differentiated X-01 hesc line by withdrawal of bfgf. The relative expression levels of RNA were quantified by qrt-pcr and were normalized to GAPDH; Data are represented as mean ± SEM.

4 Figure S2 related to Figure 2 (A, B) Transient transfection efficiency of linc-ror overexpressing vector, control vector, linc-ror-specific sirna and control RNA. (A) The percents of GFP positive (+) hes H1 cells under self-renewal conditions expressing vector-encoding GFP 3 days after transient transfection of linc-ror overexpressing vector and control vector. (B) The percents of FITC positive (+) hescs under self-renewal conditions 3 days after transient transfection of FITC-labeled linc-ror-specific sirna (siror) or

5 negative control (NC) RNA. (C-F) Relative mrna and protein levels of OCT4, SOX2, and NANOG in linc-ror-overexpressing or knockdown hes X-01 cells. (C-D) Relative mrna and protein levels of OCT4, SOX2, and NANOG in hesc X-01 under self-renewal (C) or differentiation (D) conditions that were transfected with linc-ror-overexpressing vector (linc-ror) or control vector (vector). The GFP-positive hescs were isolated by FACS. (E-F) Relative mrna and protein levels of OCT4, SOX2, and NANOG in hescs under self-renewal (E) or differentiation (F) conditions that were transfected with sirna targeting linc-ror (siror) or negative control RNA (NC). RNA and protein levels were assayed by quantitative RT-PCR and western blotting analysis; GAPDH is the normalization control. Data are represented as mean ± SEM. **, p<0.01, n=3. (G) Effort of linc-ror on the OCT4 promotor reporter in hescs. The luciferase reporters containing OCT4 promotor were co-transfected with the shown linc-ror sirna or linc-ror overexpressing vectors and their controls in the self-renewal or differentiated H1 cells. The relative luciferase activities were assayed 48 hrs after transfection and normalized to untreated self-renewal hescs. Data are represented as mean ± SEM.

6 Figure S3 related to Figure 3 (A) The kinetic expression levels of mirnas in differentiatied hescs. The relative expression levels of mirnas in hescs were quantified by qrt-pcr and

7 normalized to U6 snrna 3 days or 7 days after differentiation by the withdrawal of bfgf. Data are represented as mean ± SEM. (B) The binding ability of linc-ror, OCT4, SOX2 and NANOG full-length transcripts to Argonaute 2 (Ago2) protein. HEK293 cells were transfected with vectors expressing linc-ror, OCT4, SOX2 or NANOG full-length transcripts combined with MS2bs elements and co-transfected them into HEK293 cells with an MS2BP-YFP expression vector and a mixture of these micrornas: mir-145, mir-181a, mir-34a and mir-16. MS2BS-Renilla luciferase (RL) was used as a negative control. The transcript-specific binding RNA-protein complexes were then immunoprecipitated with YFP antibody, and IgG was used as a negative control. The immunoprecipitated proteins were assayed by Western Blotting with Argonaute 2 (Ago2) antibody. (C) mirna expression profiling upon knockdown and overexpressin of linc-ror and Oct4 knockdown. The relative expression levels of mirnas in hescs were quantified by qrt-pcr and normalized to U6 snrna 3 days after transfection of linc-ror specific sirna (siror), Oct4 specific sirna (sioct4) or linc-ror overexpressing vector (linc-ror) with scramble negative control (NC) RNA or blank vector as negative controls, respectively. Data are represented as mean ± SEM. (D) The influence of mir-145 to linc-ror expression levels. hescs H1 were transfected with mir-145 mimics. HEK293 cells were transfected with mir-145 mimics combined with vectors over-expressing (OE) linc-ror full-length transcripts. NC RNA was used as a negative control. The relative expression levels of linc-ror

8 were determined with qrt-pcr 24 hrs or 48hrs after transfections. Data are represented as mean ± SEM. ** p < 0.01, n=3. (E-G) The target validation using luciferase reporters in self-renewal hescs H1. (E) The relative luciferase activities of luciferase reporters (Luc-) containing linc-ror (RoR), OCT4, NANOG or SOX2 transcripts were assayed 48 hrs after co-transfection with the indicated micrornas or scramble NC RNA. Luc-control, the basal luciferase reporter without inserts. Data are represented as mean ± SEM. ** p < 0.01, n=3. (F) Co-expression of wild-type linc-ror (RoR WT) rescued the relative luciferase activities of luciferase reporters containing OCT4, NANOG and SOX2 when co-transfected with mir-145 in self-renewal hescs H1. Blank vector (vector) and mutant linc-ror (RoR-Mut) were used as controls. Data are represented as mean ± SEM. ** p < 0.01, n=3. (G) The self-renewal H1 cells were transfected with luciferase reporters containing wild-type (WT) or mutant (Mut) transcripts and then changed to bfgf-removing medium (-bfgf). The relative luciferase activities of cells were assayed 3 days after transfection with transfected self-renewal H1 as control (+bfgf). Data are represented as mean ± SEM. ** p < 0.01, n=3.

9 Figure S4 related to Figure 4 (A-B) linc-ror suppresses mature mir-145 expression in hesc X-01 line. The expression levels of mature mir-145 and its primary (pri-) or pre-mature (pre) transcripts in self-renewal hesc X-01 transiently transfected with wildtype (WT) linc-ror or mutant (Mut) linc-ror overexpressing vectors (A) or linc-ror-specific sirna (siror) (B). Blank vector or negative control RNA (NC) was used as controls. The relative expression levels of RNA were all quantified by qrt-pcr and were normalized to GAPDH. Data are represented as mean ± SEM. ** p < 0.01, n=3. (C) Copy numbers of linc-ror and mir-145 in two hesc lines under self-renewal or differentiated state. The exact copy numbers of primary transcripts of mir-145 (pri-mir-145), mature mir-145 and linc-ror transcripts per cell in hescs H1 and X-01 were quantified with real-time quantitative RT-PCR. The hescs H1 and X-01 were cultured under

10 self-renewal condition or differentiated condition (diff) by removing bfgf from medium. For exact quantification of gene copies per cell, linc-ror or pri-mir-145 expressing vector and reverse-transcribed mir-145 cdna were used as standard templates to formulate standard curves with limit dilution approaches, and then the exact copies of linc-ror, pri-mir-145 and mir-145 per cell were calculated according to their molecular weight and cell counts. Data are represented as mean ± SEM. ** p < 0.01, * p < 0.05, n=3.

11 Figure S5 related to Figure 5 The role of linc-ror in the maintenance of core TFs and hesc self-renewal (A) The relative mrna or mirna levels in LV-shROR infected X-01 cells referring to LV-NC infected cells. Expressions were confirmed with qrt-pcr and GAPDH or U6 snrna were used as the normalization controls. Data are represented as mean ± SEM. ** p < 0.01, n=3. (B) The cell morphology and alkaline phosphatase (AP) activity quantified by the total areas of AP positive (AP+) clones for LV-shROR-infected X-01 cells and control

12 cells under self-renewal conditions. The rescue effect of mir-145 inhibitor (inh) was also shown with negative control (NC) RNA as a control. The scale bar represents 100 μm. Data are represented as mean ± SEM. ** p < 0.01, n=3. (C) The apoptosis rates in linc-ror knock-down hescs. The apoptosis rates were assayed by apoptosis marker annexin V flow cytometry in lentivirus expressing shrna targeting linc-ror and vector-encoding GFP (LV-shROR) or negative control shrna (LV-NC) infected hescs. Lentivirus expressing shrna targeting OCT4 (LV-shOct4) was used as a positive control. The rescue effect of mir-145 inh are also shown with NC RNA as a control. Data are represented as mean ± SEM. ** p < 0.01, n=3. (D) The expression levels of differentiation markers for the three germinal layers in LV-shROR or LV-NC infected X-01 cells and mir-145 inh rescued cells were confirmed with qrt-pcr. Data are represented as mean ± SEM. ** p < 0.01, ref. to LV-NC infected cells, n=3. (E) Comparison of the expression levels of differentiation markers for the three germinal layers in LV-shROR infected hescs and mir-145 minics transfected hescs. The expression levels of differentiation markers for the three germinal layers in LV-shROR or LV-NC infected hescs, mir-145 or NC RNA transfected hescs were confirmed with qrt-pcr 3 days after treatments. Data are represented as mean ± SEM. ** p < 0.01, n=3.

13 Figure S6 related to Figure 6 (A, B) Characteristic of linc-ror-overexpressing (linc-ror OE) cells. We transfected a linc-ror-encoding lentivirus vector into ES cells, performed puromycin

14 selection to isolate linc-ror OE cells. The relative RNA levels in linc-ror OE cells referring to vector-transfected cells were assayed by qrt-pcr. GAPDH or U6 snrna were used as the normalization controls. Data are represented as mean ± SEM. **, p<0.01, n=3. (C) The cell morphology after alkaline phosphatase (AP) straining for linc-ror OE cells and vector-transfected cells under self-renewal conditions. The scale bar represents 100 μm. (D) qrt-pcr analysis for the expression levels of differentiation markers. The expression levels of differentiation markers for the three germinal layers in H1 cells transiently transfected with vectors over-expressing wildtype linc-ror or linc-ror Mut (linc-ror with the mutant mir-145 binding sites) were confirmed with qrt-pcr 3 days after transfection. Data are represented as mean ± SEM. ** p < 0.01, n=3. (E) The kinetic expression levels of core TFs and mir-145 in linc-ror knock-down cells. The kinetic expression levels of core TFs mrnas and mir-145 transcripts in LV-shROR transiently transfected hescs or control vector-transfected hescs were quantified by qrt-pcr and normalized to GAPDH or U6 snrna under differentiation conditions. Data are represented as mean ± SEM. ** p < 0.01, n=3.

15 Supplemental Table S1 related to Figure 3 Table S1 Predicted mirnas targeting OCT4, NANOG, SOX2 or linc-ror mirna name Positions of mirna binding sites OCT4 NANOG SOX2 linc-ror hsa-mir-145-5p ; hsa-mir-181a-5p ; hsa-mir-99b-3p hsa-mir-34a-5p ; hsa-mir-30c-1/2-3p hsa-mir-19b-2-5p ; hsa-mir-19a-5p to 1521 hsa-mir-125a-3p hsa-let-7a , hsa-mir-331-3p hsa-mir-205-5p hsa-mir-520c-3p Table S1 legend MiRNAs that target the full-length transcripts of OCT4, SOX2, NANOG or linc-ror were predicted using the bioinformatics tool Miranda (Enright et al., 2003). Energy Threshold: kcal/mol. The positions (ref. to the mrna start site) of these binding sites were shown.

16 Supplemental experimental procedures Cell Culture hescs (H1 and X-01) were obtained from Prof. Xiao, Zhejiang University, and cultured according to the protocol from WiCell Research Institute. Briefly, hescs were maintained in hesc culture medium on c-irradiated mouse embryonic fibroblasts (MEFs) prepared using WiCell instructions. hescs ranging from passage number were used for all of our experiments. hesc complete culture medium is composed of DMEM/F12 supplemented with 20% knockout serum replacement, 1 mm Lglutamine, 1% nonessential amino acids, 4 ng/ml human FGF2 (all from Invitrogen), and 0.1 mm 2-mercaptoethanol (Sigma). The medium was changed daily, and cells were passaged every 4-6 days with 1 mg/ml Collagen IV (Invitrogen). For differentiation studies hescs were cultured in differentiation medium (hesc medium without FGF) containing 1 mm BMP4 for 5 days, with fresh medium change daily. hescs were also maintained as feeder-free cultures on hesc qualified Matrigel (BD Biosciences) in mtesr1 medium (Stemcell Technologies) and MEF conditioned medium (CM). CM was prepared in our facility by culturing c-irradiated MEFs in complete hesc culture medium for 24 hrs, collected daily, filtered, and freezed at 220uC. FGF was added to CM before use to a final concentration of 10 ng/ml to culture the cells grown on Matrigel under pluripotent conditions. Passage 32 hescs were grown on mtesr1 medium for five passages. hescs were cultured on Matrigel following manufacturer s instructions and received fresh mtesr1 medium daily, and cells were passaged every 4-6 days with 1 mg/ml Dispase (Stem cell Technologies).

17 Differentiation by forming EB suspension was carried out in hesc medium without bfgf. Alternative differentiation method of feeder-free hescs involved the use of nonconditioned hesc medium deprived of bfgf. BMP4 differentiation was done with daily dose of 50 ng/ml BMP4 (R&D Systems) in hesc medium without bfgf for 7 days. Lentiviral Preparation and Transduction in hescs Lentiviral vectors were produced by triple transient transfection of HEK293T cells with a packaging plasmid (phr-cmvδ8.9), a plasmid encoding the envelope of vesicular stomatitis virus (VSVg) (pcmv-vsv-g) (kind gift from Prof Jia Guo, Johns Hopkins University, Shirley, MA) and the expression plasmid, employing Lipofectamine 2000 (Invitrogene). All lentivirus batches used for experiments had comparable titers ranging from to transducing functional U/ml. Virus suspensions were stored at -80 C until use and were briefly centrifuged and kept on ice immediately before use. For infection, ESCs were transduced 1 day after initial seeding of the cells with a multiplicity of infection (MOI) of 20. Cells were incubated in pluripotent maintenance media containing lentiviral particles and 4 μg/ml polybrene (Sigma Aldrich) for 18 hrs at 37 C in a humidified atmosphere containing 5% CO 2. Lentiviral particles were removed and media was replaced with fresh pluripotent maintenance media for an additional 24 hrs to permit cell recovery. Puromycin selection (1 μg/ml) started 3 days posttransduction. Experiments were carried out on the cells after at least 2 passages of continuous puromycin selection. The sequences of shrnas are depicted as follows:

18 OCT4-shRNA Sense 5'-CCGGAACAUGUGUAAGCUGCGGCCCCTCG AGGGGCCGCAGCUUACACAUGTTTTTTTG-3' AS 5'-AATTCAACAUGUGUAAGCUGCGGCCCCTC GAGGGGCCGCAGCUUACACAUGTT-3' lincror-shrna Sense 5'-CCGGTGGAGAGGAAGCCTGAGAGTCTCGA GACTCTCAGGCTTCCTCTCCTTTTTG-3' AS 5'-AATTCAAAAAGGAGAGGAAGCCTGAGAGT CTCGAGACTCTCAGGCTTCCTCTCCA-3' Dicer-shRNA Sense 5'-CCGGTAAGGGCACCCATCTCTAATTACTCG AGTAATTAGAGATGGGTGCCCTTTTTTTG-3' AS 5'-AATTCAAAAAAAGGGCACCCATCTCTAATT ACTCGAGTAATTAGAGATGGGTGCCCTTA-3' Negative control-shrna Sense 5'-CCGGTTTCTCCGAACGTGTCACGTCTCGAG ACGTGACACGTTCGGAGAATTTTTG-3' AS 5'-AATTCAAAAATTCTCCGAACGTGTCACGTC TCGAGACGTGACACGTTCGGAGAAA-3' FACS and Flow Cytometry Analysis of Self-Renewal and Apoptosis. hescs were grown in six-well plates and collected for FACS staining and quality control. Briefly, the cells were removed from the dish with trypsin/edta (Invitrogen). Trypsin was neutralized with MEF medium (DMEM containing 10% FBS [Hyclone]) and pelleted by centrifugation for 5 min at 250 g. Cells were resuspended in FACS buffer (PBS containing 2% FBS and 0.1% sodium azide). All the samples were analyzed using BD FACScalibur equipment (BD Biosciences) according to instructions from facility instrument technicians. For self-renewal analysis, each 100 ml of cell suspension ( cells) was incubated with the primary antibody mouse anti-ssea4 (Cell Signaling Technology) and PE-conjugated Goat anti-mouse (Jackson ImmunoResearch). For apoptosis analysis, cells from each sample were processed with Annexin V-PE (Annexin V-PE apoptosis detection kit, Biovision) according to the manufacturer s instructions. The cell population of interest was determined and dead cells excluded

19 using forward and side scatter parameters. Acquisition was set for 10,000 events per sample. The data were analyzed with FACSDiva software (version 4.1.2; BD Biosciences). Triplicate samples were analyzed in each experiment. Immunostaining and Fluorescent In Situ Hybridization hesc colonies were grown on Matrigel-coated coverslips in mtesr1 or MEF complete CM (CM + FGF, 10 ng/ml) or CM differentiation medium (-FGF) for 3 d. Cells were fixed with 4% paraformaldehyde in water for 15 min at room temperature. Cells were washed three times with PBS and blocked with blocking buffer (10% normal goat serum in PBS) for 2 hrs at room temperature, incubated with anti-nanog, anti-sox2, anti-oct4, anti-sox1, anti-cdx2 and anti-foxa2 antibodies (all from Abcam) overnight at room temperature, and washed twice in PBST (PBS + Tween20). The cells were then incubated with the secondary antibody (TRITC labeled goat anti-mouse IgG; Molecular Probes) for 45 min in the dark at room temperature, followed by two washes with PBST. For the detection of linc-ror, a fragment of linc-ror was amplified by the prime 5'-CCTGACCTGTTGACCCAC-3' and 5'-TTCCCAGCACCTTCTCCT-3' and then cloned in to pmd-18t vector (TAKARA). RNA probes were then transcribed in vitro with the mmassage T7 Ultra in vitro transcription kit (Ambion) and labeled with Digoxigenin (DIG)-UTP (Roche) according to the manufacture s directions. The slides were hybridized with probes overnight, washed three times with 50% formamide/2 saline-sodium citrate (SSC) and three times with 2 SSC at 45 C for 5 min each.

20 Coverslips with cells were then mounted on glass slides using Antifade mounting reagent from the Slowfade Antifade Kit (Molecular Probes). The cells were examined and photomicrographed using an Olympus fluorescence microscope. RNA Isolation and Real-Time PCR Analysis Total RNA was extracted using Trizol (Invitrogen). The mirna levels were assayed with the Taqman probes and primer sets (Applied Biosystems) according to the manufacturer s instructions. For mrna analysis, the first-strand cdna was generated using the Reverse Transcription System Kit (Promega) with random primers for RT-PCR or real-time PCR using a Power SYBR Green PCR Master Mix (Applied Biosystems) protocol in a StepOne Plus system (Applied Biosystems). U6 snrna or GAPDH mrna level was used as internal normalization control. For exact quantification of gene copies per cell, linc-ror or pri-mir-145 expressing vector and reverse-transcribed mir-145 cdna were used as standard templates to formulate standard curves with limit dilution approaches, and then the exact copies of linc-ror, pri-mir-145 and mir-145 per cell were calculated according to their molecular weight and cell counts. The primer sequences are presented as follows: linc-ror Prime S 5'-CTGGCTTTCTGGTTTGACG-3' Prime A 5'-CAGGAGGTTACTGGACTTGGAG-3' NANOG Prime S 5'-ACCTATGCCTGTGATTTGTGG-3' Prime A 5'-AGTGGGTTGTTTGCCTTTGG-3' OCT4 Prime S 5'-GAAAGCGAACCAGTATCGAGAAC-3' Prime A 5'-CCCCTGAGAAAGGAGACCCA-3' SOX2 Prime S 5'-GGTTACCTCTTCCTCCCACTCC-3' Prime A 5'-CCCTCCCATTTCCCTCGTTT-3' FOXA2 Prime S 5'-GCCGCAGATACCTCCTACTACCA-3' Prime A 5'-CCCCACTTGCTCTCTCACTTGTC-3'

21 GATA6 Prime S 5'-ATGACTCCAACTTCCACCTCTTCTAA-3' Prime A 5'-GCCCATCTTGACCCGAATACTT-3' CDX2 Prime S 5'-GTTGTTGTTGCTGCTGTT-3' Prime A 5'-CCTTCACCATATCACTTCTC-3' Brachyury Prime S 5'-CTTATTTCCGTCCATTTCCCTC-3' Prime A 5'-GCCGCAGTAGTAGTGCTGTTCT-3' MIXL1 Prime S 5'-AGCGAATTGAGATAAAGCGAGAA-3' Prime A 5'-GAGAATCACTTGAACCTGGGAG-3' NODAL Prime S 5'-CCCAAGCAGTACAACGCCTAT-3' Prime A 5'-CACCCACATTCTTCCACGAT-3' GATA4 Prime S 5'-CGGAAGCCCAAGAACCTGA-3' Prime A 5'-GCTGCTGTGCCCGTAGTGAG-3' FABP1 Prime S 5'-CAGAGCCGCAGGTCAGTCGT-3' Prime A 5'-ACCCAGCGGTGATGGTGAA-3' VIMENTEN Prime S 5'-GCCAGGCAAAGCAGGAGTC-3' Prime A 5'-AACATTGAGCAGGTCTTGGTATT-3' OTX2 Prime S 5'-CACTGCTCCAAACCCACCC-3' Prime A 5'-CAGCCCATTGACTGCGTAA-3' GATA2 Prime S 5'-GGACAGACGAAGGCAACCATT-3' Prime A 5'-GGGAAGCCAGAGGAGAAGAGG-3' HAND1 Prime S 5'-CCTATCTGGCTCTTTCTCTCTTGTC-3' Prime A 5'-CATCTTCCTGCGTCTGGTTCTC-3' RUNX2 Prime S 5'-CAGCACTCCATATCTCTACTAT-3' Prime A 5'-CTTCCATCAGCGTCAACA-3' Nestin Prime S 5'-CCCTTCCAGACTCCACTCCC-3' Prime A 5'-CCCAGCCCTCCTTTCCAG-3' PAX6 Prime S 5'-GCTGGCTGGCTTACTTCTTCCT-3' Prime A 5'-CTTTTCTCCCATTCCCACCTCT-3' SOX7 Prime S 5'-CACCAACGGGTCCCACAGA-3' Prime A 5'-GCCACTCAAGGCACAAGAAGG-3' SOX1 Prime S 5'-TCTCCAACTCGCAGGGCT-3' Prime A 5'-GGGCTCCGACTTCACCAG-3' GAPDH Prime S 5'- CGGATTTGGTCGTATTGGG-3' Prime A 5'- CTGGAAGATGGTGATGGGATT-3' Pri-miR-145 Prime S 5'-CACTCGCTCCCACCTTGTC-3' Prime A 5'-TTCTTCTTGAACCCTCATCCTG-3' Pre-miR-145 Prime S 5'-CCTTGTCCTCACGGTCCAGT-3' Prime A 5'-AACCATGACCTCAAGAACAGTATTT-3' MicroRNAs, SiRNAs and MicroRNA Inhibitor Transfection in hescs hesc colonies were grown on matrigel-coated 6-well plates. Twenty to 100 nanomolar sirnas specifically targeting linc-ror and OCT4, mir-145 minics,

22 mir-145 inhibitor or scramble control RNAs (GenePharma, Shanghai, China) microrna and 4 μl transfection reagent RNAiMAX (Invitrogene) were used to transfect each well according to the manufacturer s instructions. The sequences of sirnas are depicted as follows: OCT4-siRNA Sense 5'- AACAUGUGUAAGCUGCGGCCCdTdT -3' AS 5'- GGGCCGCAGCUUACACAUGTTdTdT -3' NANOG-siRNA Sense 5'-AAGGGUUAAGCUGUAACAUACdTdT -3' AS 5'- GUAUGUUACAGCUUAACCCUUdTdT -3' linc-ror-sirna Sense 5'- GGAGAGGAAGCCUGAGAGUdTdT -3' AS 5'- ACUCUCAGGCUUCCUCUCCdTdT -3' Dicer-siRNA Sense 5 -AAGGGCACCCAUCUCUAAUUAdTdT -3 AS 5'- TAATTAGAGATGGGTGCCCTTdTdT -3' Negative control Sense 5'-UUCUCCGAACGUGUCACGUdTdT-3' AS 5'-ACGUGACACGUUCGGAGAAdTdT-3' Vectors Construction and Transfection in hescs The complementary DNA of linc-ror, pri-mir-145, OCT4, NANOG and SOX2 was PCR-amplified from self-renewing hescs cdna by PrimeSTAR HS DNA Polymerase (TaKaRa) and was subcloned into the pcdna3.1-flag (Invitrogen), pmir-report (Ambion), pcdna3-ms2bs or pcms-egfp-vector (Clontech) to generate overexpressing or reporter vectors. Promoter of OCT4 was PCR-amplified from human genomic DNA by TaKaRa LA Taq (TaKaRa) and was subcloned into the pgl3-basic (Promega) to generate reporter vector. Mutated constructs containing the 6-8 point mutations in the seed sequence were synthesized with a QuikChange II Site-Directed Mutagenesis kit (Stratagene). In HEK293 cells, transfections of plasmids were performed using the Lipofectamine 2000 (Invitrogen) according to the manufacturer s instructions. When hescs were used, the hescs were split at 1 to 2 ratios in 24-well or 6-well plates coated with

23 matrigel. Fugene HD reagent (Promega) was used according to the manufacturer s instructions. All of the primer sequences are presented as follows: Linc-RoR For pcdna3.1-flag, pmir-report, pcdna3-ms2bs, pcms-egfp pri-mir-145 Prime S Prime A 5'-GGGGTACCGGTGAAATAAACAGCCATGTTG CTCACA-3' 5'-GGAATTCTTTATTTTTTGAGGAACTGTCATA CCGTTTCC-3' Prime S 5'-GGGGTACCAGAAGGCCAAGGCTCAGGGG-3' For pcdna3.1-flag Prime A 5'-GGAATTCTGGAAAGAAAAGCAACGCAAAG G-3' OCT4 Prime S 5'-GGAATTCCTTCGCAAGCCCTCATTTCAC-3' For pmir-report, Prime A 5'-CGCGGATCCCAGTTTGAATGCATGGGAGAG pcdna3-ms2bs C-3' OCT4 promotor Prime S 5'-GGAATTCGGTAGCTGTGTAATTGGCACCAT-3 For pgl3-basic ' Prime A 5'-CGCGGATCCAACGAACCGTCGCCAGCAA-3' NANOG Prime S 5'-GGAATTCGGAATTCCACCAGTCCCAAAGGC For AAAC-3' pmir-report Prime A 5'-CGCGGATCCCTCATTGAAACACTCGGTGAA pcdna3-ms2bs AT-3' SOX2 Prime S 5'-GGAATTCCTGCCTCTTTAAGACTAGGACTG For -3' pcdna3-ms2bs Prime A 5'-CGCGGATCCTCGGCAGACTGATTCAAATAAT ACA-3' SOX2 Prime S 5'-GGAATTC CCCCTGTGGTTACCTCTTCC-3' For pmir-report Prime A 5'-CGCGGATCCGCTGTCATTTGCTGTGGGTG-3' Luciferase Reporter Transfection and Dual Luciferase Assay HEK293 cells ( ) were seeded into each well of 96-well plate and incubated overnight then co-transfected with 80 ng reporters or mutant reporters (pmir-report), 8 ng internal control prl-tk Renilla luciferase plasmid and indicated microrna minics molecules (final concentration, 50 nm) with 1 μl of Lipofectamine For hescs, 200 ng pmir-report vectors and 20 ng prl-tk plasmid were transfected into hescs in 24-well plate with 2 μl of Lipofectamine Lysates were harvested

24 24 hrs or 48 hrs after transfection, and reporter activity was measured with the Dual Luciferase Assay (Promega). In the promoter activity assay, hescs were transfected with 200 ng of the pgl3 reporter vector, 20 ng of the Renilla control with 1.2 μl of Fugene HD reagent (Promega). The lysates were processed as described above with the Dual Luciferase Assay (Promega). Data were normalized by dividing firefly luciferase activity with that of Renilla luciferase as reported. Western Blotting Analysis Total cell lysates were prepared in a 1 sodium dodecyl sulfate buffer. Identical quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The following antibodies were used for Western blotting: anti-nanog, anti-oct4, anti-sox2 (all from Abcam), anti-dicer1 and anti-ago2 (both from Santa Cruz Biotechnology) with anti-gapdh (Santa Cruz Biotechnology) as endogenous control. Chromatin Immunoprecipitation (ChIP) Assay ChIP assays were performed according to the manufacturer s instructions of EZ-Magna ChIP A/G Kit (Millipore). Chromatin was immunoprecipitated using anti-nanog, anti-oct4 and anti-sox2 (all from Abcam) with control total human IgG (Santa Cruz). ChIP-derived DNA was quantified using realtime PCR with SYBR Green incorporation (Applied Biosystems). The promoter (pro) region of linc-ror was analyzed according to the previous report and information from UCSC websites. Three pairs of primers were designed and the best one was chose to detect the enriched genomic DNA fragments. Fold enrichments were calculated from the

25 apparent IP efficiency (ratio of ChIP enriched DNA over control IgG input DNA) and normalized to the level at a control region. The primer sequences are as follows: linc-ror pro-1 Prime S 5'- AACTGATGACATTCCACCACAAA-3' Prime A 5'-CAGTCAGCGAAGGGAGATAGG-3' linc-ror pro-2 Prime S 5'-TTTGCTTCTTCGATTCCTCCAT-3' Prime A 5'-TGTGTGGTCTTTATCTGCCTGTT-3' linc-ror pro-3 Prime S 5'-CATCCCCCTGCTATGGACG-3' Prime A 5'-GGAATGCCTTCGCCCTGT-3' linc-ror pro ref Prime S 5'-GTGGTGGAATGTCATCAGTTAAGGCG-3' Prime A 5'-TCTAGGAGTCCACCTCATAAGCAC-3' Control Prime S 5'-GAGGTCTCGTATTTGCTGCATCGTA-3' Prime A 5'-GCTAATTTCCTTCTCCACCCCAACCA-3' RNA-Binding Protein Immunoprecipitation (RIP) Assay The MS2bp-MS2bs based RIP assay was performed according to previously reports with modifications for using the EZ-Magna RIP Kit (Millipore). Briefly, HEK293 cells ( ) were seeded into 100 mm plate and incubated overnight then co-transfected with 20 μg MS2bs-cDNA overexpressing vectors (pcdna3-ms2bs) or blank control vectors with Renilla luciferase inserts (pcdna3-ms2bs-rl), 5 μg MS2bp-YFP overexpressing plasmid and indicated microrna minics molecules (final concentration, 500 nm for each minics) with 100 μl of Lipofectamine After 24 hrs, cells were crosslinked using 1% formaldehyde for 10 min at 25 C and subsequently quenched with 0.25M glycine for 5 min at room temperature. Then cells were lysed with buffer provided in kits and sonicated six times for 30 s to facilitate lysis. Immunoprecipitation was performed using anti-gfp (cross-reacting with YFP) or control IgG (both from Santa Cruz Biotechnology) according to the manufacturer s instructions. The complexes of RNA and co-isolated RNA-binding proteins were then treated with Trizol (Invitrogen) for further purification and analysis.