Data for Dermaseptin B2’s Anti-Proliferative Activity and down Regulation of Anti-Proliferative, Angiogenic and Metastatic Genes in Rhabdomyosarcoma RD Cells in Vitro
This data was obtained from the anti-proliferative activity of of Dermaseptin B2 (Drs B2) and Doxorubicin against Rhabdomyosarcoma (RD) and Vero cells and gene expression analysis of MYC, GFGFR1, NOTCH1, and CXCR7. Treatments were performed through time intervals of 24, 48, and 72-hours. Cell viability data can be obtained by using resazurin reduction assay at 24, 48, and 72-hours intervals. Gene expression data can be obtained by analyzing relative gene expression of MYC, FGFR1, NOTCH1 and CXCR7 at treatment intervals of 24, 48 and 72-hours by comparing treated and treated cells and normalization to beta-acting housekeeping reference gene using qTRPCR with Luna universal qPCR master mix.
Steps to reproduce
RD and Vero cells can be trypsinased, counted with hemocytometer and plated into 96-well cell culture plates at seeding density of 1x104 cells/well and incubated. The cells can be treated with 100µL of increasing concentrations (3, 6, 9, 12 and 15µM) of Drs B2 in complete media in each well in triplicates. Doxorubicin used as standard control drug was prepared the same way. Plates can be incubated for 24, 48, 72-hours to determine the anti-proliferative activity of Drs B2 compared to Doxorubicin. After 24, 48, 72-hours of incubation, 20µL (0.15 mg/mL) of resazurin dye can be added and incubated for 4-hours. The optical density of the cultures can then be read with plate reader (Infinite M1000, Tecan), at an absorbance of 570 nm and 600 nm. The percentage cell viability can be calculated from the net absorbance of 570 nm and 600 nm readings using the equation; percentage cell viability (% viability) = (Net absorbance of the treated cells/ Net absorbance of the blank) x 100. The effect of the Drs B2 on the proliferation of the cells can be represented in graphs of % cell viability against the log concentration (µM) of the treatment. The 50% inhibitory concentration (IC50) of the treatments can be calculated using non-linear regression analysis in GraphPad Prism software. Exponentially dividing RD cells can be treated with the corresponding IC50 values of Drs B2 and Doxorubicin for time-points of 24, 48, 72-hours. Total RNA extraction can be performed using DirectZol RNA Miniprep kit. cDNA can be synthesized using FIREScript RT cDNA Synthesis kit following manufacturer’s instructions. The expression levels of the target genes can be assessed using qRTPCR real time thermal cycler qTOWER3 84 GmbH (Analytik Jena). The qRTPCR reaction can be performed using Luna® Universal qRTPCR Master Mix (NEBM3003S, New England Biolabs) following manufacturer’s instructions. A 20 µl reaction containing 10 µl of the Luna® master mix, 0.5 µl each of the forward and reverse primers (10 pmol/ µl), 2 µl of cDNA and 7 µl of nuclease-free water. The qRTPCR can be done using the program; initial denaturation: 95◦C for 60 seconds, 40 cycles of; denaturation: 95◦C for 15 seconds, annealing/extension: 62◦C for 30 seconds, melting at 60◦C-95◦C. Data can be analyzed using Microsoft excel software. Expression levels of the target genes can be determined by using the 2^-ddCt method with reference to β-Actin housekeeping gene. The relative expression can be presented as fold change expressions relative to the controls.