Figure 3 Transfected siMDR1 inhibits the mRNA and protein expression of MDR1 in L2-RYC cells. (A) mRNA expression of MDR1 in group I, II, III, IV and IV was analyzed by real-time PCR. All cDNA samples were normalized with GAPDH. Real-time PCR results were confirmed in at least three batches of independent experiments. (*p < 0.05, vs other groups), (B) Protein expression of MDR1 was analyzed by Western blot. Protein were collected and lysed at 48 hr after treatment and MK-4827 molecular weight subjected to SDS-PAGE and Western blotting using a MDR1 antibody. Equal loading of the samples was confirmed by β-actin detection. All samples gray values were normalized with β-actin. P-glycoprotein protein relative expression of each group

was demonstrated as fold change in a histogram. (*P < 0.05, vs other groups). Analysis of P-glycoprotein activity with Daunorubicin accumulation assay Daunorubicin is a substrate of P-glycoprotein, which has red autofluorescence. Daunorubicin accumulation assay is commonly used to determine the P-glycoprotein activity [31]. We found that only cells

in group IV exhibited green fluorescence and had more visible red granular fluorescence in cytoplasm when compared with cells in other groups (Figure 4A). From flow CUDC-907 purchase cytometry data (Figure 4B and 4C), we found that red fluorescent intensity in group I, II, III and Selleckchem GDC0068 V were 70.85%, 68.42%, 70.57% and 71.72%, respectively. On the contrary, 90.85% red fluorescent positive cells were observed in group IV. Thus, our result demonstrated that siMDR1 transfected by ultrasound microbubble-mediated delivery could inhibit P-glycoprotein

function and increased intracellular Nintedanib (BIBF 1120) accumulation of Daunorubicin in L2-RYC cells. Figure 4 Daunorubicin accumulation increases in the cells treated with siMDR1-loaded Lipid microbubble transfection. The experimental groups I to V were same as that described in figure 2. L2-RYC cells were seeded in 6-well plates. Daunorubicin was added to the final concentration of 7.5 μg/ml. After 30 min, Verapamil at the final concentration of 10 μg/ml was added to terminate pumping-out of Daunorubicin. L2-RYC cells without any treatment were set as negative control. (A) Red fluorescent cells was observed under microscope, cells in group IV (cells transfected with pSEB-siMDR1s showed green fluorescent indicated by white arrow with thin arrowhead) exhibited more red granular fluorescence in cytoplasm(indicated by white arrow), (B) Red fluorescent cells were sorted by flow cytometry, (C) The percentage of red fluorescent cells of different treated groups was displayed in a histogram. (*P < 0.05, vs other groups). Sensitivity to chemotherapeutic drugs by MTT assay Next, MTT assay was also performed to determine cell viability of L2-RYC cells in vitro. Vincristine and Dactinomycin are two commonly used chemotherapeutic drugs and also substrates of P-glycoprotein.