Specific sirna

CRC cell lines HT-29 and Colo205 and fibroblast cell line CCD-18Co from the colon were purchased from ATCC. Human normal colon mucosal epithelial cell line NCM460 was obtained from INCELL. HT-29 and Colo205 were cultured in complete Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), with essential supplementation including penicillin/streptomycin solution, while NCM460 in complete RPMI-1640 medium. CCD-18Co was cultured in ATCC-formulated Eagle's Minimum Essential Medium (#30-2003) with 10% FBS. All the cells were incubated at 37°C in a 5% CO₂ humidity atmosphere. The recombinant human IL-6 was purchased from R&D Systems. For the function blocking, the specific antibody 10D5 against integrin β6 was purchased from Merck Millipore and neutralizing anti-IL-6 as well as anti-IL-6R from R&D Systems. To knock down integrin β6 (#144659), IL-6R (#106147), or STAT-3 (#116558), cells were transfected with specific siRNA (Thermo Fisher Scientific) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. For the inhibition of the STAT-3 pathway, specific STAT-3 inhibitors Stattic and Cryptotanshinone (CTS), ERK/MAPK inhibitor U0126, and PI3K inhibitor LY294002 were purchased from SelleckChem.

Specific siRNA was used to knockdown MT1F and MT1M expression (Thermo Scientific). MCF7 (approximately 50,000 cells) was transfected with MT1F and MT1M siRNA seeded onto the top insert (layered with Matrigel) of an invasion chamber (BD Biosciences, Franklin Lakes, NJ, USA). The invasion chambers were then incubated at 37°C in 5% CO₂ for 20 h. Cells that did not invade through the Matrigel (on the upper surface of the insert membrane) were mechanically removed with cotton tip applications and several washes with PBS. Invaded cells on the bottom of the coated membranes were visualized using a fluorescence microscope with a × 20 objective after incubation with Hoechst stain (Life Technologies). Images were obtained from four standardized non-overlapping fields. Invaded cells were counted using the Image J software (http://rsbweb.nih.gov/ij/). Invasion assays were done in triplicate; images of four fields per well (covering about 85% of the well) were taken for counting invaded cells. Cellular proliferation was assayed using CellTiter-Glo® Luminescence Kit (Promega, Madison, WI, USA) according to the manufacturer’s directions as described earlier [43 (link)].

All cell lines: MCF7 (HTB-22), SKBR3 (HTB-30), MDA-MB-231 (HTB-26), MDA-MB-468 (HTB-132), and H1299 (CRL-5803) were purchased from the American Type Culture Collection (ATCC, Manassas, United States) and cultivated according to the supplier’s documentation. Transient transfection with siRNA was performed with GenMute siRNA Transfection Reagent (SignaGen, Frederick, MD, USA), according to the manufacturer’s manual, using 40 nM of specific siRNA (Thermo Fisher Scientific, Waltham, MA, USA): control—(AM4613), MDM2–siMDM2#1 (s8630), siMDM2#2 (AM51334), BRCA1–siBRCA1 (s459), and RAD51–siRAD51 (VHS40453). Cells were seeded on 10-cm-diameter culture dishes (2,400,000 cells/plate) 24 h before first transfection. Transfection was performed twice, 24 h apart. Gene silencing efficiency was verified with RT-PCR and western blot analysis. Transient transfection with plasmids was performed with Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific), according to the manufacturer’s manual. Cells were treated with indicated concentrations of doxorubicin, camptothecin, etoposide, olaparib (all Selleckchem, Houston, TX, USA), and neocarzinostatin (Sigma-Aldrich, St. Louis, MO, USA).

Overexpression of MiD51 in MIN6 cells and primary mouse islet cells was achieved using the expression vector pcDNA3.1. Cells were transfected with pcDNA3.1(–)MiD51 4xMycHisx6 vector (Plasmid #44598, Addgene, Cambridge, MA) for 48 h using Effectene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Empty vector transfection served as control. MiD51 was downregulated in MIN6 cells and primary mouse islet cells by specific siRNA (Thermo Fisher, Waltham, MA, USA). A negative control siRNA (Thermo Fisher) was used for comparison. Cells were incubated for 48 h using Interferin transfection reagent (Polyplus, Illkirch-Graffenstadey, France).

For investigating a function of ZFP281, specific siRNA against Zfp281 (4390771[s105405], Thermo Fisher Scientific) was injected to IVF embryos at 6 hpi, and the injected embryos were used for NT-ETR as described above. Pre-designed negative control siRNA (RNAi Inc.) was used as a control. The NT embryos were further cultured in KSOM medium containing 0.1 μg/ml demecolcine for 24 hours, and four NT embryos were subjected to quantitative RT-PCR (qRT-PCR) using following primers: Kti12-specific primers (5′-agtctcagcctctagcctctgg-3′ and 5′-tggcctgatccaactggtg-3′), Rnd3-specific primers (5′-aagcggaacaaatcgcagag-3′ and 5′-gctaggcatgtgcgaaatcc-3′), Plk2-specific primers (5′-ctgtgtaatgtatacgatgctgctagg-3′ and 5′-tttgtggtttcgaatggaggt-3′), Aqp3-specific primers (5′-gctgtgaccttcgcaatgtg-3′ and 5′-cagcttgatccagggctctc-3′), Zfp281-specific primers (5′-cgttaccagacgtagttgggc-3′ and 5′-atgccaagtgagccacctg-3′) and 28S rRNA-specific primer (5′-aaggctaaataccggcacga-3′ and 5′-tcttaacggtttcacgccct-3′). PCR reactions were performed under the following condition using 7300 Real Time System (Thermo Fisher Scientific): 95°C 30 s; 40 cycles of 95°C, 5 s; 60°C, 31 s with the dissociation process. Two-tailed Student’s t-test was used to assess statistical significances. A P value < 0.05 was considered as statistically significant.

Preliminary set-up experiments were performed using a fluorescent siRNA (Invitrogen, Waltham, MA, USA ) and a commercial kit (RNAiMAX, Invitrogen) to find the optimal transfection conditions for rMSCs and hMSCs. The efficiency of transfection was evaluated using fluorescence-activated cell sorting (FACS) and fluorescence microscopic analyses.
After 24 h of transfection with 5 μL lipofectamine following the manufacturer’s instructions, the highest fluorescent signals were detected using the concentration of 25 nM siRNA for both rMSCs and hMSCs (Supplementary Figure S2).
Using this concentration, we transfected rMSCs (1 × 10⁶/flask) with lipofectamine and a specific siRNA (Invitrogen) to block CD73 expression. We also transfected hMSCs (1 × 10⁶/flask) with lipofectamine with or without CD73 siRNA. CD73 expression was evaluated using FACS analyses at different time points after the transfection procedure. As shown in Supplementary Figure S3A, CD73 expression was downregulated in CD73 siRNA-transfected hMSCs at 24 h after the procedure. After 48 h, CD73 expression was further decreased in both hMSCs and rMSCs transfected with siRNA (Supplementary Figure S3B,C).
Once the conditions that permit CD73 downregulation were established, EVs were collected from transfected and naïve hMSCs and rMSCs, as described below.