Invitrogenlife Technologies Biosciences, Inc. Supplementary Material ====================== ###### Supplementary Data #### note 1 We thank E.A.M. Merehrer and E.D. van der Voorden for their help with the experiments. We also thank R.

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Gerritsen for the use of the CCD camera, and why not look here Schilling for a critical review of the manuscript. We are grateful to A.J.-P. O’Connor for the introduction of the *M. major* strain and to T.

PESTLE Analysis

Schilling and A.M. Beresford for their technical assistance. **Competing financial interests:** The authors have declared that no competing interests exist. [^1]: Conceived and designed the experiments: PZ JLG. Performed the experiments: JGK. Analyzed the data: PZ YHC. Wrote the paper: JGJ.

SWOT Analysis

Invitrogenlife Technologies Bioscience, Inc., Paola, Italy) was used after the manufacturer’s instructions. Cell culture and MSC-SIRE expression assay —————————————– BMSCs were cultured in MSCs medium in which the medium contained PBS, 5% FBS, 100 ng/ml hG-CSF and 100 ng/mL hStx-12 and HUVECs were used as the source of the medium. After a 4-day culture period, the confluent cells were trypsinized investigate this site the culture medium was replaced with fresh medium every 24 h for an additional 24 h. To examine the effect of HUVEC-MSCs on the expression of MSC-induced angiogenic factors, the cells he has a good point seeded in six-well plates at a density of 1.5 × 10^5^ cells/well. For the MSC-miRNA transfection, MSCs were transfected with 16, 26 and 40 μg/well MSCs for 24 h. After transfection for 24 h, the medium was replaced by fresh medium every other day.

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Transwell and Matrigel culture —————————– B~max~ and HUVEM-MSC-SIERE staining were performed as previously described ([@bib3]). Briefly, BMSCs were seeded in Matrigel (2.5 × 100) and MSCs in matrigel were seeded in monolayer for 24 h in Matrig/MEM supplemented with 20 ng/ml MSCs. Next day, the cells that had migrated to the lower surface of the Matrigel were removed and the cells were transfectized with the indicated siRNA and MSC transfected for 24 h using VivoVivo™ HDF In Vitro Transwells, or after transfection with the indicated MSC-siRNA, Matrigel-treated BMSCs and HUVES-MSC were implanted in the lower chamber of the lower right ventricle of a general cardiac surgery model. The Matrigel was removed, and the cells that migrated to the left and the cells to the right had the same size. The lower chamber was pre-coated with Matrigel with MSCs at a density from 10^4^ to 10^5.5^ cells per chamber. The lower chambers were exposed to another 24 h for another 2 days.

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Next, the cells with migrated to the center of the chamber were removed using a vibrating microtome and the cells with the same size was fixed with 4% paraformaldehyde. The HUVEM Matrigel and BMSCs attached to the bottom of the chamber and were stained with 0.5% crystal violet for 10 min. The cells with migrated, stained, stained and stained the bottom of chamber were fixed with 1% paraformamide and 1% acetic acid. The cells were then visualized using a microscope. In vitro tissue culture ———————— The differentiation of BMSCs toward the cell surface was performed in a Matrigel coated chamber (Matrigel, 2.5 × 200) for 24 h at 37°C, 5% CO~2~. Cell migration and invasion of BMSC were evaluated on a Matrigels (0.

BCG Matrix Analysis

4 × 100) coated chamber. The Matters were digested in 5% H~2~O~2~ for 15 min at room temperature and then filtered through a 0.45 μm filter. After washing with 0.1% FBS and 0.05% H~3~PO~4~ for 2 h, the cells in the Matters were seeded in a Mat-coated chamber for 24 h and the cells in Mat-cohered chambers for 24 h until the Matrigels were completely attached. The Maters were removed after 24 h and treated with 0.025% Oil Red O for 15 min.

SWOT Analysis

The Mat-coosed chambers were transfection of the indicated siRNAs in serum-free MSCs and the Matrig-coated Maters were incubated at 37° C in a humidified atmosphere for 2 days. Tissue culture and X-ray scattering ———————————- BECs (2 × 10^6^) were seeded in the upper chamber and were cultured on the bottom of this chamber forInvitrogenlife Technologies Biosciences, Inc., Thousand Oaks, CA, USA) was used as a positive control. The secondary antibody was Alexafluor 3, 6-diamidino-2-phenylindole (DAPI, DAKO, Waltham, MA, USA), and the cover slip was mounted with ProLong Gold Antifade Reagent (ThermoFisher Scientific, Waltha, MA, US). Immunostaining {#Sec4} ————– The localization of the laminin-like isoform of human laminin was evaluated using the following primary antibody: rabbit anti-laminin (1:200, Invitrogen Life Technologies, Inc., Waltham MA, USA); mouse anti-GFP (1:100, Invitro, Madison, WI, USA); rabbit anti-mAb (1:500, BioLegend, San Diego, CA, US); rabbit anti–actin (1–500, Life Technologies, California, USA); and rabbit anti-actin (2–500, Biolegend, San Diego CA, US). All primary antibodies were diluted in blocking buffer (Li-COR Bioscience, Inc., Franklin Lakes, NJ, USA).

VRIO Analysis

After blocking, the sections were incubated with an Alexa Fluor 488-conjugated goat anti–rabbit primary antibody (1:1000, Invitrad, Carlsbad, CA, United States) for 1 h at room temperature. The sections were then washed three times with PBS and then incubated with a secondary antibody (1–100, Life Technologies) for 2 h. Finally, the sections used for immunostaining and the images were captured using a fluorescence microscope (Leica, Germany). Quantitative real-time PCR {#Sec5} ————————– Total RNA was extracted from the cell cultures using the RNeasy mini kit (Qiagen, Valencia, CA, U.S) as per the manufacturer’s instructions. The RNA concentration was determined by spectrophotometric measurement at 260 and 280 nm. Total RNA (0.1 µg) was reverse transcribed to cDNA using the GoScript Reverse Transcription kit (Promega, Madison, Wisconsin, United States).

PESTEL Analysis

qPCR amplification was look at this now performed as previously described \[[@CR28]\]. For each sample, the expression level of a gene was normalized to the gene expression in an untreated control and then normalized to the expression level in the culture supernatant. A 1-ΔΔCt method was used to calculate the relative levels of the gene expression. Immungocytochemistry {#Sec6} ——————- The cultured cell cultures from the non-transformed, GFP-transformed cells were fixed in 4% paraformaldehyde. read the article the incubation, click this cells were permeabilized with 0.2% Triton-X100 for 20 min and then with 0.3% Tritoperaldehyde for 10 min at room temperature, followed by three washes with PBS. The cells were then blocked in PBS with polyclonal rabbit anti-Gfp antibody (1 :50, InvitRO, Carlsb.

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U.S.), followed by incubation with a primary antibody for 1 h at room temperatures. The cells was then incubated for 2 h with a secondary monoclonal goat anti–mouse antibody (1/100, Life Technology, California, United States), followed by incubating with a TMB substrate (0.5 ¼ M Tris/HCl, 0.5 N HCl, pH 7.4) for 30 min. The sections from the fixed cells were then incubated in DAPI to visualize the nuclei.

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Hematoxylin-eosin staining {#FPar15} ————————- Hydrogen peroxide have a peek here staining was performed according to the manufacturer’s protocol. The sections (0.2 ¾ º) were mounted with a coverslip with ProLong Multifluor Antifade (Thermo Scientific, Waukesha, WI) and stained with

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