siRNA transfections, 2-pmol siMPS1, (5’ TTGGACTGTTATACTCTTGAA3’, SI00071624, siMiR21 (GeneSolution siRNA cat: 1027416 (mix of 4 validated anti Hs_MiR21)) (Qiagen Inc., Germantown, MD) was complexed with RNAi Max lipid transfection reagent (Invitrogen) in DMEM media for 15 minutes at ambient temperature.For NMS-P715 mediated inhibition of MPS1, cells were plated Overnight prior to drug treatment and treated with NMS-P715 at the concentrations indicated in each experiment.Proteins for RPPAt analysis was harvested 24 and 48 hours post siRNA transfection or Drug treatments
Growth protocol
U251, U87 (National Cancer Institute Frederick Tumor Repository) human GBM cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS), and maintained at 37°C, 5% CO2.
Extracted molecule
protein
Extraction protocol
Treated GBM cell (U251,U87) lysates were prepared in RPPA lysis buffer [1% Triton X-100, 50 nmol/L Hepes (pH 7.4), 150 nmol/L NaCl, 1.5 nmol/L MgCl2, 1 mmol/L EGTA, 100 nmol/L NaF, 10 nmol/L NaPPi, 10% glycerol, 1 nmol/L phenylmethylsulfonyl fluoride, 1 nmol/L Na3VO4, and aprotinin 10 μg/mL) as described elsewhere, and sent to RPPA Core Facility, MD Andersen Cancer Center, Houston, TX for RPPA analysis.
Label
DAB
Label protocol
visualized by DAB colorimetric reaction
Hybridization protocol
5 serial dilutions of lysates were arrayed on nitrocellulose‐coated slides, probed with (172 phosphorylated and non- phosphorylated) antibodies. Samples were probed with antibodies by a tyramide amplification approach (Dako CSA) and visualized by DAB colorimetric reaction.
Scan protocol
Slides were scanned on a flatbed scanner to produce 16-bit tiff images. Spots from tiff images were identified and the density was quantified by Array-Pro Analyzer software.
Description
protein lysate siNeg U87-b
Data processing
Relative protein levels for each sample were determined by interpolation of each dilution curves from the standard curve antibody slide. All the data points were normalized for protein loading and transformed to a linear value. Linear values were transformed to Log2 value and then median‐centered for hierarchical cluster analysis.
