Supplementary methods-
RNA isolation, reverse transcription and quantitative RT-PCR (qRT-PCR): Samples were prepared, run and analyzed as previously described (Youland et al 2013, J Neurooncol. 2013: 111(1): 11-18; doi: 10.1007/s11060-012-0986-1). Briefly, the RNA samples from three independent biological replicates were isolated by RNeasy (Qiagen). Reverse transcription was performed to yield cDNA. PCR mixtures containing the custom made oligodeoxynucleotide primers (IDT) for Wee1 (Forward: CGGCTCTGGAGAATTTGGTTCTGT, and Reverse: ATC AACAGAGCCCGCCAATGGCTTT) or the endogenous control β-actin (Forward: CAAGAGATGGCCACGGCTGCT, and Reverse: TCCTTCTGCATCCTGTCGGCA) were added to 384-well plates and run on an ABI Prism 7900. Relative mRNA levels of Wee1 were calculated by the SDS RQ 1.3 software (ABI).
Legends to Supplementary Figures-S1 to S5
Supplementary Figure S1: Relative quantification of Wee1 transcript in GBM xenograft lines. Total cDNA from 3 biological replicates of a tumor line were analyzed by qRT-PCR and relative abundance of Wee1 transcript normalized to β-Actin in each line.
Supplementary Figure S2: DNA damage analysis in GBM6 and GBM22 after MK-1775 treatment. A-B) shown in panel of images are the cell nuclei stained to detect gH2AX foci (green) formation, DAPI (blue) or merged in GBM6 (A) and GBM22 cells (B) at 48 h after a single treatment of 300 nM MK-1775 or radiation treatment (RT, 10 Gy). C) Quantification of cells containing both punctate and pan-nuclear gH2AX or just pan-nuclear gH2AX staining, shown in bar graph is the mean ± SEM from 3 independent experiments, * indicates p<0.05 of control versus MK-1775 treatments in each line.
Supplementary Figure S3: Growth inhibition in response to graded concentrations of MK-1775, TMZ or their combination assessed by neurosphere formation- A) GBM6, B) GBM12 and C) GBM22 cells. Neurospheres were counted 14 days after treatment; results are the mean ± SEM from 3 independent experiments and * indicates p<0.05 of comparison between TMZ/control vs. TMZ/control + MK-1775 treatments.
Supplemental Figure S4: MK-1775 therapy in GBM12 orthotopic and flank xenograft models. A. Mice with established orthotopic GBM12 xenografts were treated with placebo, TMZ 50 mg/kg once daily, and/or 50 mg/kg MK-1775 twice daily on Days 1-5, 29-31 and 57-61. The time to reach a moribund state is plotted. Early deaths were attributable to tumor burden since treatment was initiated only a few days prior to placebo mice reaching a moribund state. B) In the same experiment, mice were treated with Radiation (RT: 2 Gy/day) ± MK-1775 (Days 1-5 and 8-12) and time to reach a moribund state is plotted separately. C. Mice with established GBM12 flank xenografts were treated with placebo or TMZ 50 mg/kg and/or MK-1775 50 mg/kg twice daily on Days 1-5, 29-31 and 57-61, and the time to exceed 1500 mm3 is plotted.
Supplemental Figure S5: MALDI-TOF MS analyses of MK-1775 (m/z 501.2); A) MALDI-TOF MS of brain and flank GBM22 samples treated with placebo or a single dose of 200 mg/kg MK-1775 and harvested 2 hours later. Mass spectra from the solution standard (top), brain section (middle), and flank section (lower) are shown for (A) placebo-treated (B) MK-1775-treated mice. Results are representative of 2-3 tumor samples each. C) MALDI-TOF MS/MS of MK-1775 (m/z 501.2) in solution (top mass spectra) and in a mouse brain (bottom mass spectra) section with GBM22 tumor with a 2-hours single dose of MK-1775.