Figure 2. Microarray data analysis for gene expression in osteosarcomas upon MYC inactivation and reactivation by the StepMiner algorithm.

(A) Quantitative Real-time PCRs for MYC Transgene Expression (Human cMYC) upon treatments and removals of doxycycline in Bone Tumor Cells. Bone tumor cells 1325 were treated with 20ng/ml of doxycycline for 0, 4, 8, 12, 18, 24, 36 and 48 hours before collecting total RNA. Excess amount of PBS was applied to cells treated with doxycyline for 48 hours to remove doxycycline. At 4, 8, 12, 18, 24, 36 and 48 hours after removing doxycycline, cells were collected for total RNA by Trizol and cDNA was synthesized by Superscript II. cDNA templates from all these samples were used for quantitative real-time PCR with fluorescence labeled human c-MYC and mouse GAPDH probes to validate MYC expression. MYC expression at each time point was normalized with the MYC expression in the original tumor before doxycycline treatment. (B) Raw data from the microarray experiments at different time points was applied with StepMiner analysis to find the patterns of interest (expression went down upon MYC inactivation and stayed down upon MYC reactivation, expression went up upon MYC inactivation and stayed up upon MYC reactivation). p<0.01 was set to be the cutoff for significant changes among experiments. (C) Examples of StepMiner analysis are shown. In left panels, x-axis is the array number (MYC inactivation for 0 hour = 1, 4 hour = 2, 8 hour = 3, 12 hour = 4, 18 hour = 5, 24 hour = 6, 36 hour = 7, 48 hour = 8, MYC reactivation for 4 hour = 9, 8 hour = 10, 12 hour = 11, 18 hour = 12, 24 hour = 13, 36 hour = 14, 48 hour = 15. ) and y-axis is the level of mRNA expression compared with reference RNA. Typical one step changes are listed for Atp11a and Dnmt2 (p = 2.97×10⁻⁶ and p = 1.25×10⁻⁴ expression went up upon MYC inactivation and stayed up for MYC reactivation). Centering the data with the expression level at the identified step was applied for illustration purposes.