Real-time polymerase chain reaction Total RNA was isolated from HeLa-S3 cells by Trizol® Reagent (Life Technologies), and reverse transcription was carried out using the

Applied Biosystems High Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s instructions. The cDNA was diluted to a final concentration of approximately 1 ng/μl and reacted with gene-specific primer pairs and Applied Biosystems SYBR® Green PCR Master Mix (Life Technologies) according to the manufacturer’s protocol. The primer sequences for GAPDH (NM_002046) and β-actin (NM_001101) were designed by Origene (Rockville, MD, USA). Primer specificity was confirmed by Primer-BLAST developed at NCBI, and primer PCR efficiency was validated to be close to 100%. Genes of interest were click here detected and amplified by Applied Biosystems 7300 Real-Time PCR System (Life Technologies) with the following conditions: 2 min at 50°C, 10 min at 95°C, and 40 cycles of amplification at 95°C for 15 s and 60°C for 1 min, followed by melting curve analysis. Amplicons were visualized with electrophoresis on a 1.4% agarose gel to ensure the presence of a single product. The mRNA level of each gene was analyzed by the Applied Biosystems Sequence Detection Software

V1.2 (Life Technologies) and normalized to that of GAPDH. Relative gene expression was calculated by the comparative Ct (2−ΔΔct) method [31] and expressed as fold changes (x-fold) relative to the control. Statistical analysis Statistical analysis was performed on data from at least three independent experiments. Dabrafenib Significant difference relative to the control was tested using Student’s t test. Levels of significance of p < 0.05 and 0.01 were accepted

as significant and highly significant, respectively. Results and discussion Results PEI-NH-CNT suspensions PEI functionalization remarkably increased the degree of dispersibility of SWNTs and MWNTs. After being dispersed in ddH2O at 1 mg/ml and sonicated for 15 min, PEI-NH-MWNTs and PEI-NH-SWNTs can be solubilized in water and maintained in suspension form for over 6 months without further sonication (left why images, Figure 1A, B). Because agglomeration of carbon nanotubes as a result of van der Waals’ interaction tends to increase cytotoxicity [32, 33], PEI-NH-CNTs were subjected to centrifugation to remove large aggregates, and the supernatant gave a more homogeneous solution of PEI-NH-CNTs for the following studies (right images, Figure 1A, B). Figure 1 Suspension of PEI-NH-SWNTs and PEI-NH-MWNTs in water. PEI-NH-SWNTs (A) and PEI-NH-MWNTs (B) were solubilized in ddH2O at a concentration of 1 mg/ml and sonicated for 15 min (left images). Large aggregates were removed by centrifugation at 3,000 rpm for 30 min to obtain a more homogeneous suspension (right images). Morphology of PEI-NH-CNTs The morphology of PEI-NH-CNTs compared to pristine CNTs was studied by SEM and TEM.