Electron Microscopy of Cell Lines and Xenografts

For cell lines, ∼5,000 cells were plated in each well of 12-well plates and treated with the drug for 6 days. For xenografts, tumours were harvested and samples were cut into 1-mm³ pieces. The cell lines and tumour samples were processed similarly and Electron microscopy was performed at the High Resolution electron microscopy facility at MD Anderson Cancer Center as described previously65 (link). Briefly, they were fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3. Samples were then washed in 0.1 M sodium cacodylate buffer and treated with 0.1% Millipore-filtered cacodylate-buffered tannic acid. They were fixed with 1% buffered osmium tetroxide for 30 min and stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, filtrated and embedded in LX-112 medium. They were then polymerized in a 60 °C oven for approximately 3 days. Ultrathin sections were cut in a Leica Ultracut microtome (Leica, Deerfield, IL), stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a JEM 1010 TEM (JEOL, USA, Inc., Peabody, MA) at an accelerating voltage of 80 kV. Digital images were obtained at magnifications of × 5,000, × 25,000 and × 50,000 using the AMT Imaging System (Advanced Microscopy Techniques Corp, Danvers, MA).