Adherent cells were left to connect over night ahead of 18 hours hypoxic or normoxic preincubation and then treated with the indicated medications under normoxia or hypoxia maintained for 72 hours. By the end of the experiment, cells were subjected to SRB assay or resazurin assay. For SRB analysis, all media were eliminated and replaced with 100 l 10% trichloroacetic acid for 1 hour and washed with PBS, and fixed cells were stained with 0. Four to five SRB for 15-minutes and then washed with one of the acetic acid. Stained protein was then resuspended with 100 d 1. 5 M Tris HCl, and OD540 was measured utilizing a 96 well plate reader. For resazurin analysis, hypoxic cells were re oxygenated for 2 48 hours and then incubated with resazurin solution at 37 C for 3 hours before measurement of resorufin fluorescence. Cell survival was expressed as percent of vehicle treated control. Evaluation of apoptosis. Cell suspensions were centrifuged and mobile pellets fixed in formalin for half an hour at room temperature. Pellets were resuspended in ProLong Gold Antifade with DAPI. Apoptotic nuclear morphology was based on examining cells under UV Lymph node illumination. The percentage of apoptotic cells was determined as the average of 2 independent analyses of no less than 100 cells. Analysis of cyst spheroids. HCT116 cells were put in agarose painted 10 cm2 length meals at 2 105 cells/ml for 72 hours. Spheroids ranging from 70 to 100 m in diameter were selected and placed in spinner flasks maintained at 37 C and five minutes CO2 and allowed to achieve 500 m in diameter before incubation with ABT 737 for 24-hours at the IC20 or IC90 concentrations derived from monolayer culture studies. Spheroids were then formalin fixed and cut in to 4 m sections. Sections were deparaffinized and rehydrated, then microwaved in citrate buffer. After PBS wash, areas were blocked for 60 minutes. Sections were incubated overnight at 4 C with major antibodies against CC3 and GLUT 1. After further washes, goat anti rabbit Alexa Fluor 568 or donkey anti mouse Alexa Fluor 488 was applied price Bosutinib for 2 hours, accompanied by straight PBS washes. Slides were viewed using either a 10 or 20 goal and pictures captured. qRT PCR. Total RNA was isolated utilizing an RNeasy Kit. RNA was eluted and quantified using a Nanodrop spectrometer. The reverse transcription step was done using the TaqMan Reverse Transcription Reagent Kit based on the manufacturers guidelines. TaqMan real time PCR was designed utilizing the Universal Probe Library. Succinate dehydrogenate complex An and actin were selected as housekeeping genes. RT PCR was done with 20 ng theme cDNA using TaqMan Master Mix and an ABI Prism 7900HT sequence detection system. Cells were subjected to hypoxia or normoxia for 3 hours, after which it cycloheximide was added for 3 minutes.