Inhibition of DNA nanotube-conjugated mTOR siRNA on the growth of pulmonary arterial smooth muscle cells

Here we provide raw and processed data and methods behind mTOR siRNA loaded DNA nanotubes (siRNA-DNA-NTs) in the growth of pulmonary arterial smooth muscle cells (PASMCs) under both normoxic and hypoxic condition, and also related to (You et al., Biomaterials, 2015, 67:137–150, [1]). The MTT analysis, Semi-quantitative RT-PCR data presented here were used to probe cytotoxicity of mTOR siRNA-DNA-NT complex in its TAE-Mg2+ buffer. siRNA-DNA-NTs have a lower cytotoxicity and higher transfection efficiency and can, based on inhibition of mTOR expression, decrease PASMCs growth both hypoxic and normal condition.


Subject area Biology
More specific subject area Nanomedicine Type of data Image, graph, figure How data was acquired MTT assay, confocal laser scanning microscopy, fluorescence-activated cell sorting Data format Raw, analyzed Experimental factors Effect of nanoparticles on the cell growth in vitro Experimental features Characterization and action of siRNA loaded DNA nanotubes Data source location Chongqing, China Data accessibility The data presented in this article and is related to [1] Value of the data The data can be referenced by investigating the action of the transfection reagents for selfassembly DNA nanoparticles' transfection to mammalian cells.
These data provides a thorough understanding of the basic cytotoxicity of self-assembly DNA nanoparticles in PASMCs.
The data can provide comprehensive analysis of the regulation of self-assembly siRNA-loaded nanotubes on PASMC growth.
1. Data, experimental design, materials and methods

Analysis of the cytotoxicity of mTOR siRNA loaded DNA nanotubes by IC50
For the half-maximal inhibitory concentration (IC50) test, 4 Â 10 3 of PASMCs (per well) were cultured in 96-well plates for 24 h, and the cells were then treated with the pure nanotubes or the 1 Â TAE-Mg 2 þ buffer at various concentration for another 48 h. The cytotoxicity of the pure nanotubes and the TAE buffer was then determined by IC50 assay.

1.2.
Optimizing ratio of X-tremeGENE siRNA transfection reagents and siRNA-DNA-NTs in the cells determined by flow cytometry Based on the procedure of X-tremeGENE siRNA Transfection Reagents, first, the complex of transfection reagent and siRNA-DNA-NTs was prepared and transfected into the cells in a 24-well plate. The used ratios of the reagent (μl) to the NTs (μg) were 10:2, 2.5:0.5, 1:0.2, respectively. Then optimizing ratio of the transfection reagent to siRNA-DNA-NTs in PASMCs was tested by flow cytometry. During this process, the transfection reagent was kept in a ratio of 0.5-8 to the siRNA-NTs by varying the siRNA-NTs concentration. Samples of at least 10,000 cells were analyzed in duplicate using a FACS Calibur flow cytometer (BD Biosciences, USA)

Cell viability and cell growth assay by MTT test
PASMCs were cultured in 96-well plates (5 Â 10 3 cells per well) for 24 h, and the cells were then treated with 50 nM mTOR siRNA loaded DNA nanotubes at various doses for 0, 12, 24, or 48 h, respectively. The cell viability and growth was then determined by MTT assay as the following instruction. After the treatment, 10 μL of MTT Reagent was added to each well. Then the plate was returned to cell culture incubator for 4 h. When the purple precipitate was clearly visible under the microscope, 100 μL of Detergent Reagent was added to all wells. The plate with cover was in the dark for 3 h and then the absorbance in each well was measured at 570 nm in a microtiter plate reader.

Analysis of mTOR expression by semi-quantitative RT-PCR
At a predetermined time after transfection, total RNA was isolated from transfected PASMCs. The transfected cells were prepared for mRNA extraction using the Trizol reagent (Invitrogen, USA) according to the manufacturer's recommendations. Semi-quantitative RT-PCR analysis of mTOR mRNA expression in the transfected cells was then performed as previously described [2,3]. All samples were run in triplicate, and the levels of mRNA in each sample were normalized to those of β-actin. The mTOR forward primer was 5 0 -CGCAGGGAAGGTGATGAGGAAT-3 0 , and the reverse primer was 5 0 -GCTAAGGAGCAGCCAGGGAGAT-3 0 . The internal-control β-actin forward primer was 5 0 -TCAGGTCATCACTATCGGCAAT-3 0 , and the reverse primer was 5 0 -AAAGAAAGGGTGTAAAACGCA-3 0 . The PCR products were separated by electrophoresis on a 1.0% agarose gel containing 0.5% ethidium bromide.

Cytotoxicity of the DNA-NTs for PASMCs
In Fig. 1, the cytotoxicity of DNA nanotube solution and the solvent (1 Â TAE-Mg 2 þ ) at various concentrations in PASMCs is represented. The IC50 of DNA nanotubes was found to be 1071.4 nM (Fig. 1A). While the IC50 of the 1 Â TAE-Mg 2 þ buffer used for the DNA-NTs was approximately 0.775 Â buffer (E930 nM). The cytotoxicity of siRNA-loaded DNA-NT complex, mainly due to its TAE-Mg 2 þ buffer, was much lower to 141 nM by the pure nanomaterials (Fig. 1B).  Fig. 2A shows the typical fluorescence images of the transfection reagent-dependent cellular uptake of the mTOR siRNA-DNA-NTs, as obtained by confocal microscopy. Here, 1:0, 1:0.25, 1:0.5, 1:1 and 1:2 ratios of siRNA (μg) to X-tremeGENE siRNA Transfection Reagent (μL) were used. The cells were incubated for 24 h with DNA-NTs containing mTOR siRNACy3 at 50 nM. The merged images show whole cells with DAPI-stained nuclei. Fig. 2B shows flow cytometry analysis of the transfection efficiency of PASMCs after incubation with various ratios of siRNA-loaded nanotubes (μg) to transfection reagent (μL) for 24 h. The 1:1 ratio of X-tremeGENE siRNA Transfection Reagent (μL) to siRNA (μg) achieved high transfection efficiency (75%) for PASMCs; it was obviously less than the normal ratio (5:1) without the DNA-NTs' assistance. Fig. 3 shows in normal condition, that cell growth inhibition of mTOR siRNA-loaded nanotubes is in a time-and dose-dependent manner using MTT method. The histogram display that the cells were treated by mTOR siRNA-loaded nanotubes at doses of 12.5, 25, 50, and 100 nM, respectively, for 0, 12, 24, and 48 h, respectively. By treatment with 50 nM of mTOR siRNA loaded DNA-NTs, the inhibition rate of cell growth was 2.4%, 11.9%, 27.3%, 34.6%, 46.8% at 12, 24, 36, 48 h, respectively. Fig. 4 shows the viability of PASMCs incubated with 50 nM of mTOR siRNA treatment for predetermined amounts of time (0, 12, 24, 48 h) by the MTT assay. Cells were first cultured in 1% serum for 16 h and were then transfected with 50 nM of mTOR siRNA treatment, followed by exposure to hypoxia for another 0, 12, 24, or 48 h. Specifically, the PASMCs were pre-cultured with 3-MA  (5 mM) for 30 min and then transfected with the siRNA-DNA-NTs. MTT assay showed that the rate of cell growth decreased by 11%, 42%, and 78% compared with correspondent alone hypoxic group at 12, 24, 48 h, respectively.

Competing interests
The authors declare that they have no competing interests