The metastatic potential of the UACC-SARC1, HFF, SYO-1, and HS-SY-II cell lines were evaluated using the BD BioCoat Matrigel Invasion Chamber in duplicate (BD Biosciences, San Jose, CA) with wells prepared per manufacturer specifications. 2.5×104 cells each were added to the Matrigel Basement Membrane Matrix inserts and control inserts in duplicate and incubated for 24 hours at 37°C. Non-invading cells were removed and the membrane stained with the Hema 3 Staining System (Fisher HealthCare, Houston, TX). Counting of invading cells was facilitated by photographing the membrane though the microscope. Results were quantified as the percent invasion through the Matrigel relative to the migration through the control membrane. All images were counted by a single investigator (WZ). The data was then analyzed using Prism Graph Pad Version 6 (La Jolla, CA) to plot bar graphs and compare cell lines using a one-way ANOVA test for non-parametric data to compare the mean percent invasion for each cell line.
Graph pad prism version 6
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GraphPad Prism version 6.04 is a software application designed for data analysis, curve fitting, and scientific graphing. It provides tools for researchers to analyze and visualize their experimental data.
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7 protocols using graph pad prism version 6
1
Evaluating Metastatic Potential of Sarcoma Cell Lines
2
miRNA-mRNA Regulatory Associations
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Spearman’s correlation analysis was used to identify correlations between miRNAs and mRNA targets by ranking the expression values of miRNAs and mRNAs within datasets and calculating the correlation based on these ranks in order to discern potential regulatory associations30 (link). One proportion test was performed for each of the miRNAs as we previously described. The predicted targets that were significant by one proportion test were used for IPA. The one proportion test was calculated by significantly inversely correlated targets subtracted from total significantly correlated targets over the total significantly correlated mRNA targets (https://www.medcalc.org/calc/test_one_proportion.php ). Statistical analyses are performed using Prism GraphPad version 6 (GraphPad Software, Inc., La Jolla, CA). The alpha value < 0.05 was considered statistically significant.
3
Statistical Analysis and Imaging Quantification
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Graph-pad prism version 6.04 was used for statistical analysis and graph preparation. Student’s t test was used for pairwise comparisons. For multiple comparisons, one-way and two-way ANOVA was used. Please note that statistical details are found in the figure legends. For confocal image analysis, ZEN-Black software was used, and ImageJ was used for quantification of western blots.
4
BMP2-Induced Luciferase Assay in Cells
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LLC-PK1 cells, or C2C12 cells stably transfected with a BRE-luciferase reporter plasmid90 , were plated in complete DMEM supplemented with 10% FBS at a density of 5 × 104 cells/ml in a 96-well plate (100 μl/well) (Nunc-Immuno™ MicroWell™ 96 well polystyrene plates, Sigma-Aldrich). After 24 h LLC-PK1 cells were transfected using Lipofectamine 2000 transfection reagent (Lifetechnologies) according to manufacturer’s protocol, with 40 ng pGL3 BRE-Luciferase plasmid91 , 30 ng Renilla control plasmid and, where indicated, 20 ng of empty pHLSec vector control or test constructs as indicated. Eight hours post-transfection the cells were washed with PBS (100 μl) and serum starved in complete DMEM supplemented with 0.1% FBS overnight. Cells were stimulated with 6 nM, 10 nM or 25 nM BMP2 as indicated or buffer. Where soluble proteins were directly added to the cells, BMP2 was pre-incubated with a dilution series (from 0.4–100 × the molar concentration of BMP2) of eBMPR1A or eRGMB. After 48 hours incubation, cells were washed with PBS, lysed, and luciferase activity measured using a dual luciferase assay system (Promega) according to the manufacturer’s instructions. Luminescence was quantified using a luminometer (Tecan, Infinite 200 PRO). Graphs were produced and statistical tests carried out using GraphPad Prism Version 6.04 (GraphPad Software, La Jolla California USA).
5
Statistical Analysis and Imaging Quantification
6
DENV Infection in Wolbachia-Treated Mosquitoes
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The number of DENV infected and uninfected tissues were compared between treatment groups in each of the two replicate experiments (A and B) using Fisher’s exact test. DENV RNA copy numbers between treatment groups in each of the two replicate experiments (A and B) were compared using Mann Whitney test. Treatments were only compared within mosquito lines. Differences in Wolbachia density between treatment groups in each of the two replicate experiments (A and B) were compared using Mann Whitney test. All statistical tests were carried out in Graphpad prism Version 6.04 (San Diego, California, USA).
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BMP2-Induced Luciferase Assay in Cells
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